Intestinal carriage of extended-spectrum beta-lactamase (ESBL) -producing bacteria in food-producing animals and contamination of retail meat may contribute to increased incidences of infections with ESBL-producing bacteria in humans. Therefore, distribution of ESBL genes, plasmids and strain genotypes in Escherichia coli obtained from poultry and retail chicken meat in the Netherlands was determined and defined as 'poultry-associated' (PA). Subsequently, the proportion of E. coli isolates with PA ESBL genes, plasmids and strains was quantified in a representative sample of clinical isolates. The E. coli were derived from 98 retail chicken meat samples, a prevalence survey among poultry, and 516 human clinical samples from 31 laboratories collected during a 3-month period in 2009. Isolates were analysed using an ESBL-specific microarray, sequencing of ESBL genes, PCR-based replicon typing of plasmids, plasmid multi-locus sequence typing (pMLST) and strain genotyping (MLST). Six ESBL genes were defined as PA (bla(CTX-M-1) , bla(CTX-M-2) , bla(SHV-2) , bla(SHV-12) , bla(TEM-20) , bla(TEM-52) ): 35% of the human isolates contained PA ESBL genes and 19% contained PA ESBL genes located on IncI1 plasmids that were genetically indistinguishable from those obtained from poultry (meat). Of these ESBL genes, 86% were bla(CTX-M-1) and bla(TEM-52) genes, which were also the predominant genes in poultry (78%) and retail chicken meat (75%). Of the retail meat samples, 94% contained ESBL-producing isolates of which 39% belonged to E. coli genotypes also present in human samples. These findings are suggestive for transmission of ESBL genes, plasmids and E. coli isolates from poultry to humans, most likely through the food chain.

, a cluster of cases of pneumonia of unknown etiology were reported linked to a market in Wuhan, China 1. The causative agent was identified as the species Severe acute respiratory syndrome-related coronavirus and was named SARS-CoV-2 (ref. 2). By 16 April the virus had spread to 185 different countries, infected over 2,000,000 people and resulted in over 130,000 deaths 3. In the Netherlands, the first case of SARS-CoV-2 was notified on 27 February. The outbreak started with several different introductory events from Italy, Austria, Germany and France followed by local amplification in, and later also outside, the south of the Netherlands. The combination of near to real-time whole-genome sequence analysis and epidemiology resulted in reliable assessments of the extent of SARS-CoV-2 transmission in the community, facilitating early decision-making to control local transmission of SARS-CoV-2 in the Netherlands. We demonstrate how these data were generated and analyzed, and how SARS-CoV-2 whole-genome sequencing, in combination with epidemiological data, was used to inform public health decision-making in the Netherlands. Whole-genome sequencing (WGS) is a powerful tool to understand the transmission dynamics of outbreaks and inform outbreak control decisions 4-7. Evidence of this was seen during the 2014-2016 West African Ebola outbreak when real-time WGS was used to help public health decision-making, a strategy dubbed 'precision public health pathogen genomics' 8,9. Immediate sharing and analysis of data during outbreaks is now recommended as an integral part of outbreak response 10-12. Feasibility of real-time WGS requires access to sequence platforms that provide reliable sequences, access to metadata for interpretation, and data analysis at high speed and low cost. Therefore, WGS for outbreak support is an active area of research. Nanopore sequencing has been employed in recent outbreaks of Usutu, Ebola, Zika and yellow fever virus owing to the ease of use and relatively low start-up cost 4-7. The robustness of this method has recently been validated using Usutu virus 13,14. In the Netherlands, the first COVID-19 case was confirmed on 27 February and WGS was performed in near to real-time using an amplicon-based sequencing approach. From 22 January, symptomatic travelers from countries where SARS-CoV-2 was known to circulate were routinely tested. The first case of SARS-CoV-2 infection in the Netherlands was identified on 27 February in a person with recent travel history to Italy and an additional case was identified one day later, also in a person with recent travel history to Italy. The genomes of these first two positive samples were generated and analyzed by 29 February. These two viruses clustered differently in the phylogenetic tree, confirming separate introductions (Fig. 1a). The advice to test hospitalized patients with serious respiratory infections was issued on 24 February and subsequent attempts to identify possible local transmission chains triggered testing for SARS-CoV-2 on a large scale in h...

BackgroundResistance to carbapenem antibiotics is emerging worldwide among Enterobacteriaceae. To prevent hospital transmission due to unnoticed carriage of carbapenemase producing micro-organisms in newly admitted patients, or follow-up of patients in an outbreak setting, a molecular screening method was developed for detection of the most prevalent carbapenemase genes; blaOXA-48, blaVIM, blaIMP, blaNDM and blaKPC.MethodsA real-time multiplex PCR assay was evaluated using a collection of 86 Gram negative isolates, including 62 carbapenemase producers. Seven different laboratories carried out this method and used the assay for detection of the carbapenemase genes on a selection of 20 isolates.ResultsBoth sensitivity and specificity of the multiplex PCR assay was 100%, as established by results on the strain collection and the inter-laboratory comparisons.ConclusionsIn this study, we present a multiplex real-time PCR that is a robust, reliable and rapid method for the detection of the most prevalent carbapenemases blaOXA-48, blaVIM, blaIMP, blaNDM and blaKPC, and is suitable for screening of broth cultured rectal swabs and for identification of carbapenemase genes in cultures.

Worldwide, resistance of Gram-negative micro-organisms to third-generation cephalosporins and carbapenems owing to β-lactamases is an increasing problem. Although the CTX-M, TEM and SHV extended-spectrum β-lactamases (ESBLs) are most widely disseminated, other β-lactamase families have also recently emerged, such as plasmid-mediated AmpC β-lactamases and carbapenemases. Here we describe a new set of multiplex polymerase chain reactions (PCRs) with one amplification protocol enabling detection of 25 prevalent β-lactamase families, including ESBLs, carbapenemases, plasmid-mediated AmpC β-lactamases and OXA β-lactamases.

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