Multiple-Locus Variable Number Tandem Repeat Analysis of Staphylococcus Aureus: Comparison with Pulsed-Field Gel Electrophoresis and spa-Typing

(MRSA) is required to study the routes and rates of transmission of this pathogen. Currently available typing techniques are either resource-intensive or have limited discriminatory ability. Multiple-locus variable number tandem repeat analysis (MLVA) may provide an alternative high throughput molecular typing tool with high epidemiological resolution.-sequence typing and PFGE, at the MLVA complex level with group separation values of 95.1% and 89.2%. MLVA could not discriminate between pig-related MRSA strains isolated from humans and pigs, corroborating the high degree of relationship. MLVA was also superior in the grouping of MRSA isolates previously assigned to temporal-spatial clusters with indistinguishable SpaTypes, demonstrating its enhanced epidemiological usefulness. that yields discrete and unambiguous data that can be used to assign biological meaningful genotypes and complexes and can be used for interlaboratory comparisons in network accessible databases. Results suggest that MLVA offsets the disadvantages of other high discriminatory typing approaches and represents a promising tool for hospital, national and international molecular epidemiology

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

Plasmid diversity among genetically related Klebsiella pneumoniae bla and bla 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

Plasmid diversity among genetically related Klebsiella pneumoniae bla and bla 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

bla OXA-48-like genome architecture among carbapenemase-producing and 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

Multiple Cases of Familial Transmission of Community-Acquired Methicillin-Resistant Staphylococcus aureus

The worldwide emergence of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) can have severe public health implications. Familial transmissions of CA-MRSA in The Netherlands were investigated. Among the families studied, two clusters of CA-MRSA could be identified. This report demonstrates that family members can serve as reservoirs of CA-MRSA which may become a serious problem in containing the spread of MRSA

bla OXA-48-like genome architecture among carbapenemase-producing and 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

High prevalence of methicillin resistant Staphylococcus aureus in pigs. Vet. Microbiol

Abstract Recently methicillin resistant Staphylococcus aureus (MRSA) was isolated from pigs and pig farmers in The Netherlands. In order to assess the dissemination of MRSA in the Dutch pig population, we screened 540 pigs in 9 slaughterhouses, where a representative portion of Dutch pigs (63%) was slaughtered in 2005. We found 209 (39%) of the pigs to carry MRSA in their nares. Forty-four of 54 groups of 10 consecutive pigs (81%), each group from a different farm, and all slaughterhouses were affected. All MRSA isolates belonged to 1 clonal group, showing Multi-Locus Sequence Type 398 and closely related spa types (mainly t011, t108 and t1254). Three types of the Staphylococcal Chromosome Cassette (SCCmec) were found: III (3%), IVa (39%) and V (57%). All 44 tested isolates (1 isolate per group) were resistant to tetracycline, reflecting the high and predominant use of tetracyclines in pig husbandry. Twenty-three percent of the isolates were resistant to both erythromycin and clindamycin and 36% to kanamycin, gentamicin and tobramycin but only a single isolate was resistant to co-trimoxazole and none to ciprofloxacin and several other antibiotics. The percentage of MRSA positive pigs was significantly different among slaughterhouses and among groups within slaughterhouses, indicating a high prevalence of MRSA in pigs delivered from the farms as well as cross contamination in the slaughterhouses.

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