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.

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.

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.

cfr and fexA genes in methicillin-resistant Staphylococcus aureus from humans and livestock in the Netherlands.

Background: Although the Netherlands is a country with a low endemic level of methicillin-resistant Staphylococcus aureus (MRSA), a national MRSA surveillance has been in place since 1989. In 2003 livestock emerged as a major reservoir of MRSA and currently livestock-associated MRSA (clonal complex CC398) make up 25% of all surveillance isolates. To assess possible transfer of resistant strains or resistance genes, MRSA obtained from humans and animals were characterized in detail. Methods: The sequenced genomes of 6327 MRSA surveillance isolates from humans and from 332 CC398 isolates from livestock-related samples were analyzed and resistance genes were identified. Several isolates were subjected to long-read sequencing to reconstruct chromosomes and plasmids. Results: Here we show the presence of the multi-resistance gene cfr in seven CC398 isolates obtained from humans and in one CC398 isolate from a pig-farm dust sample. Cfr induces resistance against five antibiotic classes, which is true for all but two isolates. The isolates are genetically unrelated, and in seven of the isolates cfr are located on distinct plasmids. The fexA gene is found in 3.9% surveillance isolates and in 7.5% of the samples from livestock. There is considerable sequence variation of fexA and geographic origin of the fexA alleles. Conclusions: The rare cfr and fexA resistance genes are found in MRSA from humans and animals in the Netherlands, but there is no evidence for spread of resistant strains or resistance plasmids. The proportion of cfr-positive MRSA is low, but its presence is worrying and should be closely monitored.

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.

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.

A prospective matched case-control study on the genomic epidemiology of colistin-resistant Enterobacterales from Dutch patients.

Background: Colistin is a last-resort treatment option for infections with multidrug-resistant Gram-negative bacteria. However, colistin resistance is increasing. Methods: A six-month prospective matched case-control study was performed in which 22 Dutch laboratories with 32 associated hospitals participated. Laboratories were invited to send a maximum of five colistin-resistant Escherichia coli or Klebsiella pneumoniae (COLR-EK) isolates and five colistin-susceptible isolates (COLS-EK) to the reference laboratory, matched for patient location, material of origin and bacterial species. Epidemiological/clinical data were collected and included in the analysis. Characteristics of COLR-EK/COLS-EK isolates were compared using logistic regression with correction for variables used for matching. Forty-six ColR-EK/ColS-EK pairs were analysed by next-generation sequencing (NGS) for whole-genome multi-locus sequence typing and identification of resistance genes, including mcr genes. To identify chromosomal mutations potentially leading to colistin resistance, NGS reads were mapped against gene sequences of pmrAB, phoPQ, mgrB and crrB. Results: In total, 72 COLR-EK/COLS-EK pairs (75% E. coli and 25% K. pneumoniae) were included. Twenty-one percent of COLR-EK patients had received colistin, in contrast to 3% of COLS-EK patients (OR > 2.9). Of COLR-EK isolates, five contained mcr-1 and two mcr-9. One isolate lost mcr-9 after repeated sub-culturing, but retained colistin resistance. Among 46 sequenced COLR-EK isolates, genetic diversity was large and 19 (41.3%) isolates had chromosomal mutations potentially associated with colistin resistance. Conclusions: Colistin resistance is present but uncommon in the Netherlands and caused by the mcr gene in a minority of COLR-EK isolates. There is a need for surveillance of colistin resistance using appropriate susceptibility testing methods.

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.

MRSA surveillance programmes worldwide: moving towards a harmonised international approach.

Multinational surveillance programmes for methicillin-resistant Staphylococcus aureus (MRSA) are dependent on national structures for data collection. This study aimed to capture the diversity of national MRSA surveillance programmes and to propose a framework for harmonisation of MRSA surveillance. The International Society of Antimicrobial Chemotherapy (ISAC) MRSA Working Group conducted a structured survey on MRSA surveillance programmes and organised a webinar to discuss the programmes' strengths and challenges as well as guidelines for harmonisation. Completed surveys represented 24 MRSA surveillance programmes in 16 countries. Several countries reported separate epidemiological and microbiological surveillance. Informing clinicians and national policy-makers were the most common purposes of surveillance. Surveillance of bloodstream infections (BSIs) was present in all programmes. Other invasive infections were often included. Three countries reported active surveillance of MRSA carriage. Methodology and reporting of antimicrobial susceptibility, virulence factors, molecular genotyping and epidemiological metadata varied greatly. Current MRSA surveillance programmes rely upon heterogeneous data collection systems, which hampers international epidemiological monitoring and research. To harmonise MRSA surveillance, we suggest improving the integration of microbiological and epidemiological data, implementation of central biobanks for MRSA isolate collection, and inclusion of a representative sample of skin and soft-tissue infection cases in addition to all BSI cases.

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.

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 surveillance pilot study unveils a multicenter, clonal outbreak of VIM-2-producing Pseudomonas aeruginosa ST111 in the Netherlands between 2015 and 2017.

Verona Integron-encoded Metallo-beta-lactamase (VIM) is the most frequently-encountered carbapenemase in the healthcare-related pathogen Pseudomonas aeruginosa. In the Netherlands, a low-endemic country for antibiotic-resistant bacteria, no national surveillance data on the prevalence of carbapenemase-producing P. aeruginosa (CPPA) was available. Therefore, in 2016, a national surveillance pilot study was initiated to investigate the occurrence, molecular epidemiology, genetic characterization, and resistomes of CPPA among P. aeruginosa isolates submitted by medical microbiology laboratories (MMLs) throughout the country. From 1221 isolates included in the study, 124 (10%) produced carbapenemase (CIM-positive); of these, the majority (95, 77%) were positive for the blaVIM gene using PCR. Sequencing was performed on 112 CIM-positive and 56 CIM-negative isolates (n = 168), and genetic clustering revealed that 75/168 (45%) isolates were highly similar. This genetic cluster, designated Group 1, comprised isolates that belonged to high-risk sequence type ST111/serotype O12, had similar resistomes, and all but two carried the blaVIM-2 allele on an identical class 1 integron. Additionally, Group 1 isolates originated from around the country (i.e. seven provinces) and from multiple MMLs. In conclusion, the Netherlands had experienced a nationwide, inter-institutional, clonal outbreak of VIM-2-producing P. aeruginosa for at least three years, which this pilot study was crucial in identifying. A structured, national surveillance program is strongly advised to monitor the spread of Group 1 CPPA, to identify emerging clones/carbapenemase genes, and to detect transmission in and especially between hospitals in order to control current and future outbreaks.

A genetic cluster of MDR Enterobacter cloacae complex ST78 harbouring a plasmid containing bla VIM-1 and mcr-9 in the Netherlands.

BACKGROUND: Carbapenemases produced by Enterobacterales are often encoded by genes on transferable plasmids and represent a major healthcare problem, especially if the plasmids contain additional antibiotic resistance genes. As part of Dutch national surveillance, 50 medical microbiological laboratories submit their Enterobacterales isolates suspected of carbapenemase production to the National Institute for Public Health and the Environment for characterization. All isolates for which carbapenemase production is confirmed are subjected to next-generation sequencing. OBJECTIVES: To study the molecular characteristics of a genetic cluster of Enterobacter cloacae complex isolates collected in Dutch national surveillance in the period 2015-20 in the Netherlands. METHODS: Short- and long-read genome sequencing was used in combination with MLST and pan-genome MLST (pgMLST) analyses. Automated antimicrobial susceptibility testing (AST), the Etest for meropenem and the broth microdilution test for colistin were performed. The carbapenem inactivation method was used to assess carbapenemase production. RESULTS: pgMLST revealed that nine E. cloacae complex isolates from three different hospitals in the Netherlands differed by <20 alleles and grouped in a genetic cluster termed EclCluster-013. Seven isolates were submitted by one hospital in 2016-20. EclCluster-013 isolates produced carbapenemase and were from ST78, a globally disseminated lineage. EclCluster-013 isolates harboured a 316 078 bp IncH12 plasmid carrying the bla VIM-1 carbapenemase and the novel mcr-9 colistin resistance gene along with genes encoding resistance to different antibiotic classes. AST showed that EclCluster-013 isolates were MDR, but susceptible to meropenem (<2 mg/L) and colistin (<2 mg/L). CONCLUSIONS: The EclCluster-013 reported here represents an MDR E. cloacae complex ST78 strain containing an IncH12 plasmid carrying both the bla VIM-1 carbapenemase and the mcr-9 colistin resistance gene.

Vitek® 2 MICs as first-line phenotypic screening method for carbapenemase-producing Pseudomonas aeruginosa.

Aim: To define sensitivity and specificity of Vitek® 2 MICs as phenotypic screening method for carbapenemase-producing Pseudomonas aeruginosa. Materials & methods: We determined Vitek® 2 MICs of antipseudomonal antimicrobials in 130 unrelated carbapenemase-producing P. aeruginosa and 129 carbapenemase-negative P. aeruginosa isolates within a Dutch carbapenemase-surveillance database. We calculated test characteristics of single and combined antimicrobial MICs for carbapenemase production. Results: Vitek® 2 MIC above epidemiological cutoff of both imipenem and tobramycin or ciprofloxacin and tobramycin displayed a sensitivity of 96.2% and specificity of 89.6% for carbapenemase production in P. aeruginosa. Conclusion: Vitek® 2 MIC> epidemiological cut-off values seem sensitive and specific as a phenotypic screening strategy for carbapenemase-producing P. aeruginosa. Combining imipenem and tobramycin or ciprofloxacin and tobramycin performed best as a screening strategy for defining which P. aeruginosa isolates should undergo confirmatory tests for carbapenemase production.

blaOXA-48-like genome architecture among carbapenemase-producing Escherichia coli and Klebsiella pneumoniae in the Netherlands.

Carbapenem-hydrolysing enzymes belonging to the OXA-48-like group are encoded by blaOXA-48-like alleles and are abundant among Enterobacterales in the Netherlands. Therefore, the objective here was to investigate the characteristics, gene content and diversity of the blaOXA-48-like carrying plasmids and chromosomes of Escherichia coli and Klebsiella pneumoniae collected in the Dutch national surveillance from 2014 to 2019 in comparison with genome sequences from 29 countries. A combination of short-read genome sequencing with long-read sequencing enabled the reconstruction of 47 and 132 complete blaOXA-48-like plasmids for E. coli and K. pneumoniae, respectively. Seven distinct plasmid groups designated as pOXA-48-1 to pOXA-48-5, pOXA-181 and pOXA-232 were identified in the Netherlands which were similar to internationally reported plasmids obtained from countries from North and South America, Europe, Asia and Oceania. The seven plasmid groups varied in size, G+C content, presence of antibiotic resistance genes, replicon family and gene content. The pOXA-48-1 to pOXA-48-5 plasmids were variable, and the pOXA-181 and pOXA-232 plasmids were conserved. The pOXA-48-1, pOXA-48-2, pOXA-48-3 and pOXA-48-5 groups contained a putative conjugation system, but this was absent in the pOXA-48-4, pOXA-181 and pOXA-232 plasmid groups. pOXA-48 plasmids contained the PemI antitoxin, while the pOXA-181 and pOXA-232 plasmids did not. Furthermore, the pOXA-181 plasmids carried a virB2-virB3-virB9-virB10-virB11 type IV secretion system, while the pOXA-48 plasmids and pOXA-232 lacked this system. A group of non-related pOXA-48 plasmids from the Netherlands contained different resistance genes, non-IncL-type replicons or no replicons. Whole genome multilocus sequence typing revealed that the blaOXA-48-like plasmids were found in a wide variety of genetic backgrounds in contrast to chromosomally encoded blaOXA-48-like alleles. Chromosomally localized blaOXA-48 and blaOXA-244 alleles were located on genetic elements of variable sizes and comprised regions of pOXA-48 plasmids. The blaOXA-48-like genetic element was flanked by a direct repeat upstream of IS1R, and was found at multiple locations in the chromosomes of E. coli. Lastly, K. pneumoniae isolates carrying blaOXA-48 or blaOXA-232 were mostly resistant for meropenem, whereas E. coli blaOXA-48, blaOXA-181 and chromosomal blaOXA-48 or blaOXA-244 isolates were mostly sensitive. In conclusion, the overall blaOXA-48-like plasmid population in the Netherlands is conserved and similar to that reported for other countries, confirming global dissemination of blaOXA-48-like plasmids. Variations in size, presence of antibiotic resistance genes and gene content impacted pOXA-48, pOXA-181 and pOXA-232 plasmid architecture.

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.

Plasmid diversity among genetically related Klebsiella pneumoniae blaKPC-2 and blaKPC-3 isolates collected in the Dutch national surveillance.

Carbapenemase-producing Klebsiella pneumoniae emerged as a nosocomial pathogen causing morbidity and mortality in patients. For infection prevention it is important to track the spread of K. pneumoniae and its plasmids between patients. Therefore, the major aim was to recapitulate the contents and diversity of the plasmids of genetically related K. pneumoniae strains harboring the beta-lactamase gene blaKPC-2 or blaKPC-3 to determine their dissemination in the Netherlands and the former Dutch Caribbean islands from 2014 to 2019. Next-generation sequencing was combined with long-read third-generation sequencing to reconstruct 22 plasmids. wgMLST revealed five genetic clusters comprised of K. pneumoniae blaKPC-2 isolates and four clusters consisted of blaKPC-3 isolates. KpnCluster-019 blaKPC-2 isolates were found both in the Netherlands and the Caribbean islands, while blaKPC-3 cluster isolates only in the Netherlands. Each K. pneumoniae blaKPC-2 or blaKPC-3 cluster was characterized by a distinct resistome and plasmidome. However, the large and medium plasmids contained a variety of antibiotic resistance genes, conjugation machinery, cation transport systems, transposons, toxin/antitoxins, insertion sequences and prophage-related elements. The small plasmids carried genes implicated in virulence. Thus, implementing long-read plasmid sequencing analysis for K. pneumoniae surveillance provided important insights in the transmission of a KpnCluster-019 blaKPC-2 strain between the Netherlands and the Caribbean.

Routinely available antimicrobial susceptibility information can be used to increase the efficiency of screening for carbapenemase-producing Enterobacteriaceae.

Introduction. Increased carbapenem resistance is often caused by carbapenemase production.Aim. The objective of our study was to assess which antibiotic susceptibility patterns, as tested by automated systems, are highly associated with the absence of carbapenemase production in Enterobacteriaceae isolates, and could therefore be used as a screening tool.Methodology. Routine antibiotic susceptibility testing data from 42 medical microbiology laboratories in the Netherlands in the period between January 2011 and June 2017 were obtained from the national antimicrobial resistance surveillance programme. Data on Enterobacteriaceae isolates that had an elevated minimum inhibitory concentration (MIC) for carbapenems (meropenem >0.25 mg l-1 or imipenem >1.0 mg l-1) were selected and subjected to phenotypic or genotypic carbapenemase production testing. Routinely available amoxicillin/clavulanic acid, piperacillin/tazobactam, cefuroxime and ceftriaxone/cefotaxime susceptibilities were studied in relation to carbapenemase production by calculating the negative predictive value.Results. No evidence for carbapenemase-producing Enterobacteriaceae (CPE) was found in 767 of 1007 (76 %) isolates. The negative predictive value was highest for amoxicillin/clavulanic acid (99.6 %) and piperacillin/tazobactam (98.8 %).Conclusion. Enterobacteriaceae isolates with elevated carbapenem MICs that are susceptible to amoxicillin/clavulanic acid or piperacillin/tazobactam are unlikely to be carbapenemase producers. Preselection based on this susceptibility pattern may lead to increased laboratory efficiency and reduction of costs. Whether this is also true for countries with a different distribution of CPE species and types or a higher prevalence of CPE needs to be studied.