An unusual outbreak in the Netherlands: community-onset impetigo caused by a meticillin-resistant Staphylococcus aureus with additional resistance to fusidic acid, June 2018 to January 2020.

In this retrospective observational study, we analysed a community outbreak of impetigo with meticillin-resistant Staphylococcus aureus (MRSA), with additional resistance to fusidic acid (first-line treatment). The outbreak occurred between June 2018 and January 2020 in the eastern part of the Netherlands with an epidemiological link to three cases from the north-western part. Forty nine impetigo cases and eight carrier cases were identified, including 47 children. All but one impetigo case had community-onset of symptoms. Pharmacy prescription data for topical mupirocin and fusidic acid and GP questionnaires suggested an underestimated outbreak size. The 57 outbreak isolates were identified by the Dutch MRSA surveillance as MLVA-type MT4627 and sequence type 121, previously reported only once in 2014. Next-generation sequencing revealed they contained a fusidic acid resistance gene, exfoliative toxin genes and an epidermal cell differentiation inhibitor gene. Whole-genome multilocus sequence typing revealed genetic clustering of all 19 sequenced isolates from the outbreak region and isolates from the three north-western cases. The allelic distances between these Dutch isolates and international isolates were high. This outbreak shows the appearance of community-onset MRSA strains with additional drug resistance and virulence factors in a country with a low prevalence of antimicrobial resistance.

National point prevalence study on carriage of multidrug-resistant microorganisms in Dutch long-term care facilities in 2018.

OBJECTIVES: Long-term care facilities (LTCFs) may act as a reservoir of ESBL-producing Enterobacterales (ESBL-E) and carbapenemase-producing Enterobacterales (CPE) for hospitals and the general population. In this study, we estimated the prevalence and molecular epidemiology of rectal carriage with ESBL-E and CPE in residents of Dutch LTCFs between March 2018 and December 2018. METHODS: LTCFs were geographically selected across the country. For each LTCF, a random sample of residents were tested for ESBL-E and CPE in 2018. To identify risk factors for high carriage prevalence and/or individual carriage, characteristics of LTCFs and of a subset of the tested residents were collected. WGS was conducted on isolates from LTCFs with an ESBL-E prevalence of >10% and all CPE isolates to identify institutional clonal transmission. RESULTS: A total of 4420 residents of 159 LTCFs were included. The weighted mean ESBL-E prevalence was 8.3% (95% CI: 6.8-10.0) and no CPE were found. In 53 LTCFs (33%), where ESBL-E prevalence was >10%, MLST using WGS (wgMLST) was performed. This included 264 isolates, the majority being Escherichia coli (n = 224) followed by Klebsiella pneumoniae (n = 30). Genetic clusters were identified in more than half (30/53; 57%) of high ESBL-positive LTCFs. Among the E. coli isolates, blaCTX-M-15 (92/224; 41%) and blaCTX-M-27 (40/224; 18%) were the most prevalent ESBL-encoding genes. For K. pneumoniae isolates, the most common was blaCTX-M-15 (23/30; 80%). CONCLUSIONS: The estimated prevalence of ESBL-E rectal carriage in Dutch LTCFs is 8.3% and resistance is observed mainly in E. coli with predominance of blaCTX-M-15 and blaCTX-M-27. ESBL-E prevalence in LTCFs seems comparable to previously reported prevalence in hospitals and the general population.

Outbreak of NDM-1-Producing Klebsiella pneumoniae in a Dutch Hospital, with Interspecies Transfer of the Resistance Plasmid and Unexpected Occurrence in Unrelated Health Care Centers.

In the Netherlands, the number of cases of infection with New Delhi metallo-beta-lactamase (NDM)-positive Enterobacteriaceae is low. Here, we report an outbreak of NDM-1-producing Klebsiella pneumoniae infection in a Dutch hospital with interspecies transfer of the resistance plasmid and unexpected occurrence in other unrelated health care centers (HCCs). Next-generation sequencing was performed on 250 carbapenemase-producing Enterobacteriaceae isolates, including 42 NDM-positive isolates obtained from 29 persons at the outbreak site. Most outbreak isolates were K. pneumoniae (n = 26) and Escherichia coli (n = 11), but 5 isolates comprising three other Enterobacteriaceae species were also cultured. The 26 K. pneumoniae isolates had sequence type 873 (ST873), as did 7 unrelated K. pneumoniae isolates originating from five geographically dispersed HCCs. The 33 ST873 isolates that clustered closely together using whole-genome multilocus sequence typing (wgMLST) carried the same plasmids and had limited differences in the resistome. The 11 E. coli outbreak isolates showed great variety in STs, did not cluster using wgMLST, and showed considerable diversity in resistome and plasmid profiles. The blaNDM-1 gene-carrying plasmid present in the ST873 K. pneumoniae isolates was found in all the other Enterobacteriaceae species cultured at the outbreak location and in a single E. coli isolate from another HCC. We describe a hospital outbreak with an NDM-1-producing K. pneumoniae strain from an unknown source that was also found in patients from five other Dutch HCCs in the same time frame without an epidemiological link. Interspecies transfer of the resistance plasmid was observed in other Enterobacteriaceae species isolated at the outbreak location and in another HCC.

Next-Generation Sequencing Confirms Presumed Nosocomial Transmission of Livestock-Associated Methicillin-Resistant Staphylococcus aureus in the Netherlands.

UNLABELLED: Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) was detected in 2003 and rapidly became the predominant MRSA clade in the Netherlands. Studies have shown that transmissions are difficult to identify, since this MRSA variant represents a genetically homogenous clade when current typing techniques are used. Here, next-generation sequencing was performed on 206 LA-MRSA isolates to assess the capability of LA-MRSA to be transmitted between humans. The usefulness of single nucleotide variants (SNVs), the composition of the SCCmec region, and the presence of plasmids to identify transmission of LA-MRSA were assessed. In total, 30 presumed putative nosocomial transmission events and 2 LA-MRSA outbreaks were studied; in most cases, SNV analysis revealed that the isolates of the index patient and the contact(s) clustered closely together. In three presumed events, the isolates did not cluster together, indicating that transmission was unlikely. The composition of the SCCmec region corroborated these findings. However, plasmid identification did not support our SNV analysis, since different plasmids were present in several cases where SNV and SCCmec analysis suggested that transmission was likely. Next-generation sequencing shows that transmission of LA-MRSA does occur in Dutch health care settings. Transmission was identified based on SNV analysis combined with epidemiological data and in the context of epidemiologically related and unrelated isolates. Analysis of the SCCmec region provided limited, albeit useful, information to corroborate conclusions on transmissions, but plasmid identification did not. IMPORTANCE: In 2003, a variant of methicillin-resistant Staphylococcus aureus (MRSA) isolated from pigs was also found in pig farmers in France and the Netherlands. Soon thereafter, this livestock-associated MRSA (LA-MRSA) was identified in many other countries. Transmission of LA-MRSA between humans, particularly in the health care setting, is regarded to occur sporadically. Moreover, studies that describe LA-MRSA transmission used molecular characterization of isolates with limited discriminatory power, making the validity of the conclusion that transmission occurred questionable. In our study, we sequenced the complete genomes of 206 LA-MRSA isolates, obtained from more than 30 presumed LA-MRSA transmission events. Analysis of the data showed that transmission of LA-MRSA between humans had indeed occurred in more than 90% of these events. We conclude that transmission of LA-MRSA between humans does occur in Dutch health care settings; therefore, a decision to discontinue the search and destroy policy for LA-MRSA should be taken with caution.

Changing characteristics of livestock-associated meticillin-resistant Staphylococcus aureus isolated from humans - emergence of a subclade transmitted without livestock exposure, the Netherlands, 2003 to 2014.

Since 2007, livestock-associated meticillin-resistant Staphylococcus aureus (LA-MRSA) has become the predominant MRSA clade isolated from humans in the Netherlands. To assess possible temporal changes, we molecularly characterised over 9,000 LA-MRSA isolates submitted from 2003 to 2014 to the Dutch MRSA surveillance. After an initial rapid increase with a peak in 2009 (n = 1,368), the total number of submitted LA-MRSA isolates has been slowly decreasing to 968 in 2014 and over 80% of LA-MRSA belonged to one of three predominant MLVA/spa-types. Next generation sequencing (n=118) showed that MT569/t034 isolates were genetically more diverse than MT398/t011 and MT572/t108. Concurrent with the decrease in LA-MRSA, fewer people reported having contact with livestock and this was most prominent for people carrying MT569/t034 LA-MRSA. The proportion of LA-MRSA isolated from infection-related materials increased from 6% in 2009, to 13% in 2014 and most of these isolates originated from patients older than 50 years of age. Remarkably, 83% of these patients reported not having contact with livestock. The results reveal an ongoing change in the genotypic and epidemiological characteristics of Dutch LA-MRSA isolated from humans with the emergence of a LA-MRSA subclade independent of livestock exposure, suggesting LA-MRSA starts to resemble non-LA-MRSA in terms of transmissibility and pathogenicity.

Whole-Genome Mapping as a Novel High-Resolution Typing Tool for Legionella pneumophila.

Legionella is the causative agent for Legionnaires' disease (LD) and is responsible for several large outbreaks in the world. More than 90% of LD cases are caused by Legionella pneumophila, and studies on the origin and transmission routes of this pathogen rely on adequate molecular characterization of isolates. Current typing of L. pneumophila mainly depends on sequence-based typing (SBT). However, studies have shown that in some outbreak situations, SBT does not have sufficient discriminatory power to distinguish between related and nonrelated L. pneumophila isolates. In this study, we used a novel high-resolution typing technique, called whole-genome mapping (WGM), to differentiate between epidemiologically related and nonrelated L. pneumophila isolates. Assessment of the method by various validation experiments showed highly reproducible results, and WGM was able to confirm two well-documented Dutch L. pneumophila outbreaks. Comparison of whole-genome maps of the two outbreaks together with WGMs of epidemiologically nonrelated L. pneumophila isolates showed major differences between the maps, and WGM yielded a higher discriminatory power than SBT. In conclusion, WGM can be a valuable alternative to perform outbreak investigations of L. pneumophila in real time since the turnaround time from culture to comparison of the L. pneumophila maps is less than 24 h.

Studying Bordetella pertussis populations by use of SNPeX, a simple high-throughput single nucleotide polymorphism typing method.

Large outbreaks of pertussis occur despite vaccination. A first step in the analyses of outbreaks is strain typing. However, the typing of Bordetella pertussis, the causative agent of pertussis, is problematic because the available assays are insufficiently discriminatory, not unequivocal, time-consuming, and/or costly. Here, we describe a single nucleotide primer extension assay for the study of B. pertussis populations, SNPeX (single nucleotide primer extension), which addresses these problems. The assay is based on the incorporation of fluorescently labeled dideoxynucleotides (ddNTPs) at the 3' end of allele-specific poly(A)-tailed primers and subsequent analysis with a capillary DNA analyzer. Each single nucleotide polymorphism (SNP) primer has a specific length, and as a result, up to 20 SNPs can be determined in one SNPeX reaction. Importantly, PCR amplification of target DNA is not required. We selected 38 SNPeX targets from the whole-genome sequencing data of 74 B. pertussis strains collected from across the world. The SNPeX-based phylogenetic trees preserved the general tree topology of B. pertussis populations based on whole-genome sequencing, with a minor loss of details. We envisage a strategy whereby SNP types (SnpTs) are quickly identified with the SNPeX assay during an outbreak, followed by whole-genome sequencing (WGS) of a limited number of isolates representing predominant SnpTs and the incorporation of novel SNPs in the SNPeX assay. The flexibility of the SNPeX assay allows the method to evolve along with the pathogen, making it a promising method for studying outbreaks of B. pertussis and other pathogens.

Detection and serotyping of pneumococci in community acquired pneumonia patients without culture using blood and urine samples.

BACKGROUND: Treatment of community acquired pneumonia (CAP) patients with antibiotics before laboratory-confirmed diagnosis leads to loss of knowledge on the causative bacterial pathogen. Therefore, an increasing number of pneumococcal infections is identified using non-culture based techniques. However, methods for serotyping directly on the clinical specimen remain scarce. Here we present three approaches for detection and serotyping of pneumococci using samples from patients with CAP. METHODS: The first approach is quantitative PCR (qPCR) analysis on blood samples (n = 211) followed by capsular sequence typing (CST) to identify the serotype. The second approach, a urinary antigen assay (n = 223), designated as inhibition multiplex immunoassay (IMIA), is based on Luminex technology targeting 14 serotypes. The third approach is a multiplex immunoassay (MIA) (n = 171) also based on Luminex technology which detects serologic antibody responses against 14 serotypes. The three alternative assays were performed on samples obtained from 309 adult hospitalized CAP patients in 2007-2010 and the results were compared with those obtained from conventional laboratory methods to detect pneumococcal CAP, i.e. blood cultures, sputum cultures and BinaxNOW urinary antigen tests. RESULTS: Using qPCR, MIA and IMIA, we were able to detect the pneumococcus in samples of 56% more patients compared to conventional methods. Furthermore, we were able to assign a serotype to the infecting pneumococcus from samples of 25% of all CAP patients, using any of the three serotyping methods (CST, IMIA and MIA). CONCLUSION: This study indicates the usefulness of additional molecular methods to conventional laboratory methods for the detection of pneumococcal pneumonia. Direct detection and subsequent serotyping on clinical samples will improve the accuracy of pneumococcal surveillance to monitor vaccine effectiveness.

Immunogenicity of 13-valent pneumococcal conjugate vaccine administered according to 4 different primary immunization schedules in infants: a randomized clinical trial.

IMPORTANCE: Immunization schedules with pneumococcal conjugate vaccine (PCV) differ among countries regarding the number of doses, age at vaccinations, and interval between doses. OBJECTIVE: To assess the optimal primary vaccination schedule by comparing immunogenicity of 13-valent PCV (PCV13) in 4 different immunization schedules. DESIGN, SETTING, AND PARTICIPANTS: An open-label, parallel-group, randomized clinical trial of healthy term infants in a general community in The Netherlands conducted between June 30, 2010, and January 25, 2011, with 99% follow-up until age 12 months. INTERVENTIONS: Infants (N = 400) were randomly assigned (1:1:1:1) to receive PCV13 either at ages 2, 4, and 6 months (2-4-6); at ages 3 and 5 months (3-5); at ages 2, 3, and 4 months (2-3-4); or at ages 2 and 4 months (2-4), with a booster dose at age 11.5 months. MAIN OUTCOMES AND MEASURES: Primary outcome measure was antibody geometric mean concentrations (GMCs) against PCV13-included serotypes 1 month after the booster dose measured by multiplex immunoassay. Secondary outcomes included GMCs measured 1 month after the primary series, at 8 months of age, and before the booster. RESULTS: The primary outcome, GMCs at 1 month after the booster dose, was not significantly different between schedules for 70 of 78 comparisons. The 2-4-6 schedule was superior to the 2-3-4 schedule for serotypes 18C (10.2 µg/mL [95% CI, 8.2-12.7] vs 6.5 µg/mL [95% CI, 5.4-7.8]) and 23F (10.9 µg/mL [95% CI, 9.0-13.3] vs 7.3 µg/mL [95% CI, 5.8-9.2]) and superior to the 2-4 schedule for serotypes 6B (8.5 µg/mL [95% CI, 7.1-10.2] vs 5.1 µg/mL [95% CI 3.8-6.7]), 18C (6.6 µg/mL [95% CI, 5.7-7.7]), and 23F (7.2 µg/mL [95% CI, 5.9-8.8]). For serotype 1, the 3-5 schedule (11.7 µg/mL [95% CI, 9.6-14.3]) was superior to the other schedules. Geometric mean concentrations for all 13 serotypes ranged between 1.6 and 19.9 µg/mL. Secondary outcomes demonstrated differences 1 month after the primary series. The 2-4-6 schedule was superior compared with the 3-5, 2-3-4, and 2-4 schedules for 3, 9, and 11 serotypes, respectively. Differences between schedules persisted until the booster dose. CONCLUSIONS AND RELEVANCE: The use of 4 different PCV13 immunization schedules in healthy term infants resulted in no statistically significant differences in antibody levels after the booster dose for almost all serotypes. The choice of PCV schedule will require a balance between the need for early protection and maintaining protection between the primary series and the booster. TRIAL REGISTRATION: trialregister.nl Identifier: NTR2316.

Molecular characterization of MRSA collected during national surveillance between 2008 and 2019 in the Netherlands.

BACKGROUND: Although the Netherlands is a country with a low endemic level, methicillin-resistant Staphylococcus aureus (MRSA) poses a significant health care problem. Therefore, high coverage national MRSA surveillance has been in place since 1989. To monitor possible changes in the type-distribution and emergence of resistance and virulence, MRSA isolates are molecularly characterized. METHODS: All 43,321 isolates from 36,520 persons, collected 2008-2019, were typed by multiple-locus variable number tandem repeats analysis (MLVA) with simultaneous PCR detection of the mecA, mecC and lukF-PV genes, indicative for PVL. Next-generation sequencing data of 4991 isolates from 4798 persons were used for whole genome multi-locus sequence typing (wgMLST) and identification of resistance and virulence genes. RESULTS: We show temporal change in the molecular characteristics of the MRSA population with the proportion of PVL-positive isolates increasing from 15% in 2008-2010 to 25% in 2017-2019. In livestock-associated MRSA obtained from humans, PVL-positivity increases to 6% in 2017-2019 with isolates predominantly from regions with few pig farms. wgMLST reveals the presence of 35 genogroups with distinct resistance, virulence gene profiles and specimen origin. Typing shows prolonged persistent MRSA carriage with a mean carriage period of 407 days. There is a clear spatial and a weak temporal relationship between isolates that clustered in wgMLST, indicative for regional spread of MRSA strains. CONCLUSIONS: Using molecular characterization, this exceptionally large study shows genomic changes in the MRSA population at the national level. It reveals waxing and waning of types and genogroups and an increasing proportion of PVL-positive MRSA.

A group of bacteria that cause difficult-to-treat infections in humans is methicillin-resistant Staphylococcus aureus (MRSA). The aim of this study was to monitor changes in the spread of MRSA, their disease causing potential and resistance to antibiotics used to treat MRSA infections. MRSA from patients and their contacts in the Netherlands were collected over a period of 12 years and characterized. This revealed new types of MRSA emerged and others disappeared. An increasing number of MRSA produces a protein called PVL toxin, enabling MRSA to cause more severe infections. Also, some people appear to carry MRSA without any disease for more than a year. These findings suggest an increasing disease potential of MRSA and possible unnoticed sources of infection. Consequently, it is important to maintain monitoring of these infections to minimize MRSA spread.

Markers of epidemiological success of methicillin-resistant Staphylococcus aureus 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 analysis and linear regression. Antimicrobial usage data from ESAC-Net was compared with national MRSA incidence data. RESULTS: Heterogeneity of MRSA isolate collections across countries hampered the use of a unified operational definition of success; therefore, country-specific approaches were used to establish the MACOTRA strain collection. Phenotypic antimicrobial resistance varied within related MRSA populations and across countries. In time-scaled haplotypic density analysis, fluoroquinolone, macrolide and mupirocin resistance were associated with MRSA success, whereas gentamicin, rifampicin and trimethoprim resistance were associated with sporadicity. Usage of antimicrobials across 29 European countries varied substantially, and ß-lactam, fluoroquinolone, macrolide and aminoglycoside use correlated with MRSA incidence. DISCUSSION: Our results are the strongest yet to associate MRSA antibiotic resistance profiles and antibiotic usage with the 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.

Widespread Detection of Yersiniabactin Gene Cluster and Its Encoding Integrative Conjugative Elements (ICEKp) among Nonoutbreak OXA-48-Producing Klebsiella pneumoniae Clinical Isolates from Spain and the Netherlands.

In this study, we determined the presence of virulence factors in nonoutbreak, high-risk clones and other isolates belonging to less common sequence types associated with the spread of OXA-48-producing Klebsiella pneumoniae clinical isolates from The Netherlands (n = 61) and Spain (n = 53). Most isolates shared a chromosomally encoded core of virulence factors, including the enterobactin gene cluster, fimbrial fim and mrk gene clusters, and urea metabolism genes (ureAD). We observed a high diversity of K-Locus and K/O loci combinations, KL17 and KL24 (both 16%), and the O1/O2v1 locus (51%) being the most prevalent in our study. The most prevalent accessory virulence factor was the yersiniabactin gene cluster (66.7%). We found seven yersiniabactin lineages-ybt 9, ybt 10, ybt 13, ybt 14, ybt 16, ybt 17, and ybt 27-which were chromosomally embedded in seven integrative conjugative elements (ICEKp): ICEKp3, ICEKp4, ICEKp2, ICEKp5, ICEKp12, ICEKp10, and ICEKp22, respectively. Multidrug-resistant lineages-ST11, ST101, and ST405-were associated with ybt 10/ICEKp4, ybt 9/ICEKp3, and ybt 27/ICEKp22, respectively. The fimbrial adhesin kpi operon (kpiABCDEFG) was predominant among ST14, ST15, and ST405 isolates, as well as the ferric uptake system kfuABC, which was also predominant among ST101 isolates. No convergence of hypervirulence and resistance was observed in this collection of OXA-48-producing K. pneumoniae clinical isolates. Nevertheless, two isolates, ST133 and ST792, were positive for the genotoxin colibactin gene cluster (ICEKp10). In this study, the integrative conjugative element, ICEKp, was the major vehicle for yersiniabactin and colibactin gene clusters spreading. IMPORTANCE Convergence of multidrug resistance and hypervirulence in Klebsiella pneumoniae isolates has been reported mostly related to sporadic cases or small outbreaks. Nevertheless, little is known about the real prevalence of carbapenem-resistant hypervirulent K. pneumoniae since these two phenomena are often separately studied. In this study, we gathered information on the virulent content of nonoutbreak, high-risk clones (i.e., ST11, ST15, and ST405) and other less common STs associated with the spread of OXA-48-producing K. pneumoniae clinical isolates. The study of virulence content in nonoutbreak isolates can help us to expand information on the genomic landscape of virulence factors in K. pneumoniae population by identifying virulence markers and their mechanisms of spread. Surveillance should focus not only on antimicrobial resistance but also on virulence characteristics to avoid the spread of multidrug and (hyper)virulent K. pneumoniae that may cause untreatable and more severe infections.

Invasive pneumococcal disease and 7-valent pneumococcal conjugate vaccine, the Netherlands.

In the Netherlands, the national immunization program includes 7-valent pneumococcal conjugate vaccine (PCV7) for all newborns born after April 1, 2006. We compared the incidence of invasive pneumococcal disease (IPD) and patient and disease characteristics before PCV7 introduction (June 2004-June 2006) with those after PCV7 introduction (June 2008-June 2010). Culture-confirmed IPD cases were identified by 9 sentinel laboratories covering ≈25% of the Dutch population. Significant declines in overall IPD incidence were observed in children <2 (60%) and in persons >65 (13%) years of age. A trend toward gradual increases in non-PCV7 serotype IPD infections was observed in all age groups; the largest increases were among persons 50-64 (37%) and >65 (25%) years of age. In adults, the proportion of immunocompromised persons increased among IPD patients. Overall, deaths from IPD decreased from 16% to 12% because of a lower case-fatality rate for persons with non-PCV7 serotype IPD.

Evaluation of Neisseria gonorrhoeae multiple-locus variable-number tandem-repeat analysis, N. gonorrhoeae Multiantigen sequence typing, and full-length porB gene sequence analysis for molecular epidemiological typing.

The performance characteristics of Neisseria gonorrhoeae multilocus variable-number tandem-repeat analysis were evaluated, by comparison with N. gonorrhoeae multiantigen sequence typing and full-length porB sequence typing. Assessment of the relatedness of intra- and interpatient isolates showed that all three genotyping techniques display a high resolution and typeability.

High genetic diversity of Staphylococcus aureus strains colonizing patients with epidermolysis bullosa.

Patients with the blistering disease, epidermolysis bullosa (EB), frequently suffer from chronic wounds that become colonized by pathogenic bacteria, such as Staphylococcus aureus. To determine S. aureus colonization rates in patients with EB, swabs were collected from the anterior nares, throats and wounds of 52 Dutch patients with EB. Swabs were also collected from nares and throats of 13 healthcare workers who occasionally meet the sampled patients with EB. All EB patients with chronic wounds and 75% of the patients without chronic wounds were colonized with S. aureus. In contrast, 39% of the sampled healthcare workers were colonized with S. aureus. Typing revealed a high degree of genetic diversity of 184 collected S. aureus isolates. Autoinoculation of S. aureus in individual patients with EB was shown to occur frequently, whereas transmission of S. aureus between patients with EB is apparently rare. There was no evidence for S. aureus transmission between patients with EB and healthcare workers.

Comparative analysis of IMP-4- and OXA-58-containing plasmids of three carbapenemase-producing Acinetobacter ursingii strains in the Netherlands.

OBJECTIVES: A recent occurrence of carbapenemase-producing Acinetobacter ursingii was reported in the Netherlands and comprised three unrelated strains carrying the blaIMP-4 and blaOXA-58 encoding genes. The objective was to investigate a putative common source of the carbapenemase resistance genes and plasmids in these A. ursingii strains. METHODS: Hybrid assembly of short-read and long-read sequencing data was performed using Unicycler and assembled genomes were analysed by ResFinder and PlasmidFinder. RESULTS: Hybrid assemblies of A. ursingii genomes yielded a circular chromosome, a large plasmid harboring blaIMP-4 and blaOXA-58 genes (sizes 259-317kb), and four to five other smaller plasmids. ResFinder analyses revealed 16 other acquired resistance genes on the plasmids carrying the blaIMP-4 and blaOXA-58 genes. These 18 genes encode resistance towards eight antibiotic classes. The smaller plasmids did not carry acquired resistance genes. Comparative analysis showed that the three blaIMP-4/blaOXA-58 plasmids were similar (61%-83%) and shared 13 to 17 of the 18 resistance genes. BLAST analysis showed that the blaIMP-4/blaOXA-58 plasmids were not reported before. However, a close match with a 399 kb plasmid from Acinetobacter johnsonii was found (99% similarity, 80% coverage). This A. johnsonii plasmid contains the blaOXA-58 gene, but lacks blaIMP-4, and it shares eight other resistance genes with those present on the A. ursingii blaIMP-4/blaOXA-58 plasmids. CONCLUSION: Three blaIMP-4/blaOXA-58-carrying plasmids were characterized in three carbapenemase-producing A. ursingii strains. The plasmids were highly similar, suggesting a putative common source or co-selection of resistance genes from A. johnsonii. These results provide initial insights in the dissemination of carbapenem-resistance in A. ursingii in the Netherlands.

Epidemiology of carbapenem-resistant and carbapenemase-producing Enterobacterales in the Netherlands 2017-2019.

BACKGROUND: The Netherlands is currently considered a low endemic country for carbapenem-resistant Enterobacterales (CRE) and carbapenemase-producing Enterobacterales (CPE), experiencing only sporadic hospital outbreaks. This study aims to describe susceptibility to carbapenems and the epidemiology of carbapenemase production in Enterobacterales in the Netherlands in 2017-2019. METHODS: Three complementary nationwide surveillance systems are in place to monitor carbapenem susceptibility in the Netherlands. Routine antimicrobial susceptibility test results from medical microbiology laboratories were used to study phenotypic susceptibility of Escherichia coli and Klebsiella pneumoniae. Pathogen surveillance (of all Enterobacterales species) and mandatory notifications were used to describe the characteristics of CPE positive isolates and affected persons. RESULTS: The prevalence of isolates with gradient strip test-confirmed elevated meropenem (> 0.25 mg/L) or imipenem (> 1 mg/L) minimum inhibitory concentration (MIC) in the Netherlands was very low in 2017-2019, with percentages of 0.06% in E. coli and 0.49% in K. pneumoniae, and carbapenem resistances of 0.02% and 0.18%, respectively. A total of 895 unique species/carbapenemase-encoding allele combinations of CPE from 764 persons were submitted between 2017 and 2019, with the annual number of submissions increasing slightly each year. Epidemiological data was available for 660 persons. Screening because of presumed colonisation risk was the reason for sampling in 70.0% (462/660) of persons. Hospitalization abroad was the most common risk factor, being identified in 45.9% of persons. CONCLUSIONS: Carbapenem resistance of E. coli and K. pneumoniae remains low in the Netherlands. The annual number of CPE isolates slightly increased during the period 2017-2019. Recent hospitalization abroad is the main risk factor for acquisition of CPE.

Genetic Characteristics of Methicillin-Resistant Staphylococcus argenteus Isolates Collected in the Dutch National MRSA Surveillance from 2008 to 2021.

Staphylococcus argenteus is a recently described member of the Staphylococcus aureus complex (SAC) and is associated with human disease. The frequency and intensity of infections caused by S. argenteus are similar to those of Staphylococcus aureus. S. argenteus can harbor antibiotic resistance genes and a variety of virulence factors analogous to methicillin-resistant S. aureus (MRSA). The aim of our study was to analyze a collection of isolates in the Dutch national MRSA surveillance from January 2008 until March 2021 that were nontypeable by multilocus variable-number tandem-repeat analysis (MLVA). Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-ToF MS) was used for identifying the S. argenteus isolates, and whole-genome sequencing and SeqSphere were used to generate an in-house whole-genome multilocus sequence typing (wgMLST) scheme for typing the isolates. Furthermore, the presence of antibiotic resistance genes, replicons, and virulence genes was determined. Of 52,467 isolates submitted as MRSA from January 2008 until March 2021, 64 isolates (0.12%) were nontypeable with MLVA, and 54 of them were identified with mass spectrometry (MALDI-ToF MS) as S. argenteus. It appeared in retrospect that the first methicillin-resistant S. argenteus (MRSArg) was already submitted in 2008. An in-house-developed S. argenteus wgMLST scheme revealed that S. argenteus isolates clustered in 5 genomic groups which were characterized by distinct MLST types, resistomes, plasmid replicon families, and virulence factors. All but one isolate carried the staphylococcal chromosomal cassette mec (SCCmec) type IV harboring the methicillin resistance gene mecA and represent MRSArg. Most of the isolates with SCCmec subtype IVc(2B) had a trimethoprim resistance gene, dfrG, and harbored a blaZ-carrying plasmid, and most MRSArg isolates have the immune-modulating genes scn and sak. Nine of the 47 isolates carried enterotoxin-encoding genes seg, sei, sem, seo, and seu, which might be able to cause food poisoning. In some persons there was long-term persistence of MRSArg, and there were several genetically related MRSArg isolates in people living in close proximity, suggesting direct human-human transmission. IMPORTANCE We show that MRSArg has been circulating in the Netherlands since at least 2008. Although MRSArg is distinct from MRSA, it has a comparable population structure and carries similar resistance and virulence genes. The Dutch national MRSA surveillance has been expanded to include other methicillin-resistant members of the S. aureus complex, such as S. argenteus and Staphylococcus schweitzeri.

Livestock-associated methicillin-resistant Staphylococcus aureus epidemiology, genetic diversity, and clinical characteristics in an urban region.

Objectives: While Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA), defined as CC398, is a well-known pathogen among those working with livestock, there are indications that LA-MRSA prevalence among the general population is increasing. However, the clinical impact in urban areas remains unknown. The aim of this study was to assess the genetic epidemiology and clinical characteristics of LA-MRSA in an urban area with a limited livestock population. Methods: In this retrospective study, we evaluated LA-MRSA strains that were collected between 2014 and 2018 from patients who received clinical care in a single urban area in Netherlands. Patient files were assessed for livestock exposure data, clinical findings, and contact tracing information. Next-generation sequencing (NGS) analysis in combination with wgMLST was conducted to assess genetic diversity and relatedness and to detect virulence and resistance genes. Results: LA-MRSA strains were cultured from 81 patients, comprising 12% of all the MRSA strains found in seven study laboratories between 2014 and 2018. No livestock link was found in 76% of patients (n = 61), and 28% of patients (n = 23) had an infection, mostly of the skin or soft tissue. Contact tracing had been initiated in 14 cases, leading to the identification of two hospital transmissions: a cluster of 9 cases and one of 2 cases. NGS data were available for 91% (n = 75) of the patients. wgMLST confirmed the clusters detected via contact tracing (n = 2) and identified 5 additional clusters without a known epidemiological link. Relevant resistance and virulence findings included the PVL virulence gene (3 isolates) and tetracycline resistance (79 isolates). Conclusion: LA-MRSA may cause a relevant burden of disease in urban areas. Surprisingly, most infections in the present study occurred in the absence of a livestock link, suggesting inter-human transmission. These findings and the presence of PVL and other immune evasive complex virulence genes warrant future surveillance and preventative measures.