Combining epidemiological data and whole genome sequencing to understand SARS-CoV-2 transmission dynamics in a large tertiary care hospital during the first COVID-19 wave in The Netherlands focusing on healthcare workers.

BACKGROUND: Healthcare facilities have been challenged by the risk of SARS-CoV-2 transmission between healthcare workers (HCW) and patients. During the first wave of the COVID-19 pandemic, infections among HCW were observed, questioning infection prevention and control (IPC) measures implemented at that time. AIM: This study aimed to identify nosocomial transmission routes of SARS-CoV-2 between HCW and patients in a tertiary care hospital. METHODS: All SARS-CoV-2 PCR positive HCW and patients identified between 1 March and 19 May 2020, were included in the analysis. Epidemiological data were collected from patient files and HCW contact tracing interviews. Whole genome sequences of SARS-CoV-2 were generated using Nanopore sequencing (WGS). Epidemiological clusters were identified, whereafter WGS and epidemiological data were combined for re-evaluation of epidemiological clusters and identification of potential transmission clusters. HCW infections were further classified into categories based on the likelihood that the infection was acquired via nosocomial transmission. Secondary cases were defined as COVID-19 cases in our hospital, part of a transmission cluster, of which the index case was either a patient or HCW from our hospital. FINDINGS: The study population consisted of 293 HCW and 245 patients. Epidemiological data revealed 36 potential epidemiological clusters, with an estimated 222 (75.7%) HCW as secondary cases. WGS results were available for 195 HCW (88.2%) and 20 patients (12.8%) who belonged to an epidemiological cluster. Re-evaluation of the epidemiological clusters, with the available WGS data identified 31 transmission clusters with 65 (29.4%) HCW as secondary cases. Transmission clusters were all part of 18 (50.0%) previously determined epidemiological clusters, demonstrating that several larger outbreaks actually consisted, of several smaller transmission clusters. A total of 21 (7.2%) HCW infections were classified as from confirmed nosocomial, of which 18 were acquired from another HCW and 3 from a patient. CONCLUSION: The majority of SARS-CoV-2 infections among HCW could be attributed to community-acquired infection. Infections among HCW that could be classified as due to nosocomial transmission, were mainly caused by HCW-to-HCW transmission rather than patient-to-HCW transmission. It is important to recognize the uncertainties of cluster analyses based solely on epidemiological data.

Enterobacteriaceae and Bacteroidaceae provide resistance to travel-associated intestinal colonization by multi-drug resistant Escherichia coli.

Previous studies have shown high acquisition risks of extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-E) among international travelers visiting antimicrobial resistance (AMR) hotspots. Although antibiotic use and travelers' diarrhea have shown to influence the ESBL-E acquisition risk, it remains largely unknown whether successful colonization of ESBL-E during travel is associated with the composition, functional capacity and resilience of the traveler's microbiome. The microbiome of pre- and post-travel fecal samples from 190 international travelers visiting Africa or Asia was profiled using whole metagenome shotgun sequencing. A metagenomics species concept approach was used to determine the microbial composition, population diversity and functional capacity before travel and how it is altered longitudinally. Eleven travelers were positive for ESBL-E before travel and removed from the analysis. Neither the microbial richness (Chao1), diversity (effective Shannon) and community structure (Bray-Curtis dissimilarity) in pretravel samples nor the longitudinal change of these metrics during travel were predictive for ESBL-E acquisition. A zero-inflated two-step beta-regression model was used to determine how the longitudinal change in both prevalence and abundance of each taxon was related to ESBL acquisition. There were detected increases in both the prevalence and abundance of Citrobacter freundii and two members of the genus Bacteroides, in association with remaining uncolonized by ESBL-E. These results highlight the potential of these individual microbes as a microbial consortium to prevent the acquisition of ESBL-E. The ability to alter a person's colonization resistance to a bacterium could be key to intervention strategies that aim to minimize the spread of MDR bacteria.

Destination shapes antibiotic resistance gene acquisitions, abundance increases, and diversity changes in Dutch travelers.

BACKGROUND: Antimicrobial-resistant bacteria and their antimicrobial resistance (AMR) genes can spread by hitchhiking in human guts. International travel can exacerbate this public health threat when travelers acquire AMR genes endemic to their destinations and bring them back to their home countries. Prior studies have demonstrated travel-related acquisition of specific opportunistic pathogens and AMR genes, but the extent and magnitude of travel's effects on the gut resistome remain largely unknown. METHODS: Using whole metagenomic shotgun sequencing, functional metagenomics, and Dirichlet multinomial mixture models, we investigated the abundance, diversity, function, resistome architecture, and context of AMR genes in the fecal microbiomes of 190 Dutch individuals, before and after travel to diverse international locations. RESULTS: Travel markedly increased the abundance and α-diversity of AMR genes in the travelers' gut resistome, and we determined that 56 unique AMR genes showed significant acquisition following international travel. These acquisition events were biased towards AMR genes with efflux, inactivation, and target replacement resistance mechanisms. Travel-induced shaping of the gut resistome had distinct correlations with geographical destination, so individuals returning to The Netherlands from the same destination country were more likely to have similar resistome features. Finally, we identified and detailed specific acquisition events of high-risk, mobile genetic element-associated AMR genes including qnr fluoroquinolone resistance genes, blaCTX-M family extended-spectrum ß-lactamases, and the plasmid-borne mcr-1 colistin resistance gene. CONCLUSIONS: Our results show that travel shapes the architecture of the human gut resistome and results in AMR gene acquisition against a variety of antimicrobial drug classes. These broad acquisitions highlight the putative risks that international travel poses to public health by gut resistome perturbation and the global spread of locally endemic AMR genes.

Author Correction: Global phylogenetic analysis of Escherichia coli and plasmids carrying the mcr-1 gene indicates bacterial diversity but plasmid restriction.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Prevalence and risk factors for carriage of ESBL-producing Enterobacteriaceae in a population of Dutch travellers: A cross-sectional study.

BACKGROUND: We investigated prevalence and predictive factors for ESBL-E carriage in a population of mostly travellers prior to their travel (n = 2216). In addition, we examined ESBL genotype before travel and compared these to returning travellers. METHOD: A questionnaire and faecal sample were collected before travel, and a second faecal sample was collected immediately after travel. Faecal samples were analysed for ESBL-E, with genotypic characterization by PCR and sequencing. Risk factors for ESBL-E carriage prior to travel were identified by logistic regression analyses. RESULTS: Before travel, 136 participants (6.1%) were colonized with ESBL-E. Antibiotic use in the past three months (ORadjusted 2.57; 95% CI 1.59-4.16) and travel outside of Europe in the past year (1.92, 1.28-2.87) were risk factors for ESBL-E colonisation prior to travel. Travel outside of Europe carried the largest attributable risk (39.8%). Prior to travel 31.3% (40/128) of participants carried blaCTX-M 15 and 21.9% (28/128) blaCTX-M 14/18. In returning travellers 633 acquired ESBL-E of who 53.4% (338/633) acquired blaCTX-M 15 and 17.7% (112/633) blaCTX-M 14/18. CONCLUSION: In our population of Dutch travellers we found a pre-travel ESBL-E prevalence of 6.1%. Prior to travel, previous antibiotic use and travel outside of Europe were the strongest independent predictors for ESBL-E carriage, with travel outside of Europe carrying the largest attributable risk. Our molecular results suggest ESBL genes found in our study population prior to travel were in large part travel related.

Risk of acquisition of human diarrhoeagenic Escherichia coli virulence genes in intercontinental travellers: A prospective, multi-centre study.

BACKGROUND: We studied geographic distribution of diarrhoeagenic Escherichia coli virulence genes (DEC VGs) acquisition in travellers and investigated if they acquired highly virulent EAEC/STEC hybrid strains. METHODS: From the prospective, multicentre COMBAT study among 2001 Dutch travellers, 491 travellers were selected based on travel destination to 7 subregions. Faecal samples taken directly before and after travel were screened for nine DEC VGs with real-time PCR. Incidence proportions and rates were calculated for each gene and subregion. RESULTS: 479 travellers were analysed. 21.8% acquired aggR (EAEC), with highest acquisition rates in Northern and Western Africa and 15.3% acquired eae (STEC/EPEC) with highest rates in travellers to Western and Eastern Africa. ETEC (elt or est gene) was acquired by 4.2% of travellers and acquisition of est was associated with traveller's diarrhoea. Overall, the risk of acquiring DEC VGs was low in Southern Africa and South America. Although the combination of aggR (EAEC) and stx1/2 (STEC) was acquired by 3 travellers, these genes could not be detected together in a single E. coli strain. CONCLUSIONS: The risk of acquisition of DEC VGs strongly depends on the travel destination, with those travelling to Africa - except Southern Africa - having a higher risk.

Carriage of Blastocystis spp. in travellers - A prospective longitudinal study.

INTRODUCTION: A lack of prospective and longitudinal data on pre- and post-travel carriage of Blastocystis spp. complicates interpretation of a positive test post-travel. Therefore we studied dynamics of Blastocystis carriage in a cohort of Dutch travellers. METHODS: From the prospective, multicentre COMBAT study among 2001 Dutch travellers, a subset of 491 travellers was selected based on travel destination to 7 subregions (70 or 71 travellers each). Faecal samples taken directly before and after travel were screened for Blastocystis with qPCR, followed, when positive, by sequence analysis to determine subtypes. RESULTS: After exclusion of 12 samples with missing samples or inhibited qPCR-reactions, stool samples of 479 travellers were analysed. Before travel, 174 of them (36.3%) carried Blastocystis and in most of these, the same subtype was persistently carried. However, in 48/174 of those travellers (27.6%; CI95 20.8-36.6%) no Blastocystis or a different subtype was detected in the post-travel sample, indicating loss of Blastocystis during travel. Only 26 (5.4%; CI95 3.7%-8.0%) of all travellers acquired Blastocystis, including two individuals that were already positive for Blastocystis before travel but acquired a different subtype during travel. DISCUSSION: This study shows that Blastocystis carriage in travellers is highly dynamic. The observed acquisition and loss of Blastocystis could either be travel-related or reflect the natural course of Blastocystis carriage. We demonstrate that the majority of Blastocystis detected in post-travel samples were already carried before travel.

Global phylogenetic analysis of Escherichia coli and plasmids carrying the mcr-1 gene indicates bacterial diversity but plasmid restriction.

To understand the dynamics behind the worldwide spread of the mcr-1 gene, we determined the population structure of Escherichia coli and of mobile genetic elements (MGEs) carrying the mcr-1 gene. After a systematic review of the literature we included 65 E. coli whole genome sequences (WGS), adding 6 recently sequenced travel related isolates, and 312 MLST profiles. We included 219 MGEs described in 7 Enterobacteriaceae species isolated from human, animal and environmental samples. Despite a high overall diversity, 2 lineages were observed in the E. coli population that may function as reservoirs of the mcr-1 gene, the largest of which was linked to ST10, a sequence type known for its ubiquity in human faecal samples and in food samples. No genotypic clustering by geographical origin or isolation source was observed. Amongst a total of 13 plasmid incompatibility types, the IncI2, IncX4 and IncHI2 plasmids accounted for more than 90% of MGEs carrying the mcr-1 gene. We observed significant geographical clustering with regional spread of IncHI2 plasmids in Europe and IncI2 in Asia. These findings point towards promiscuous spread of the mcr-1 gene by efficient horizontal gene transfer dominated by a limited number of plasmid incompatibility types.

Travel-related acquisition of diarrhoeagenic bacteria, enteral viruses and parasites in a prospective cohort of 98 Dutch travellers.

BACKGROUND: Limited prospective data are available on the acquisition of viral, bacterial and parasitic diarrhoeagenic agents by healthy individuals during travel. METHODS: To determine the frequency of travel associated acquisition of 19 pathogens in 98 intercontinental travellers, qPCR was used to detect 8 viral pathogens, 6 bacterial enteric pathogens and 5 parasite species in faecal samples collected immediately before and after travel. RESULTS: We found high pre-travel carriage rates of Blastocystis spp. and Dientamoeba fragilis of 32% and 19% respectively. Pre-travel prevalences of all other tested pathogens were below 3%. Blastocystis spp. (10%), Plesiomonas shigelloides (7%), D. fragilis (6%) and Shigella spp. (5%) were the most frequently acquired pathogens and acquisition of enteral viruses and hepatitis E virus in this relatively small group of travellers was rare or non-existent. CONCLUSIONS: Our findings suggest that the role of viruses as the cause of persisting traveller's diarrhoea is limited and bacterial pathogens are more likely as a cause of traveller's diarrhoea. The substantial proportion of travellers carrying Blastocystis spp. and D. fragilis before travel warrants cautious interpretation of positive samples in returning travellers with gastrointestinal complaints.

Import and spread of extended-spectrum ß-lactamase-producing Enterobacteriaceae by international travellers (COMBAT study): a prospective, multicentre cohort study.

BACKGROUND: International travel contributes to the dissemination of antimicrobial resistance. We investigated the acquisition of extended-spectrum ß-lactamase-producing Enterobacteriaceae (ESBL-E) during international travel, with a focus on predictive factors for acquisition, duration of colonisation, and probability of onward transmission. METHODS: Within the prospective, multicentre COMBAT study, 2001 Dutch travellers and 215 non-travelling household members were enrolled. Faecal samples and questionnaires on demographics, illnesses, and behaviour were collected before travel and immediately and 1, 3, 6, and 12 months after return. Samples were screened for the presence of ESBL-E. In post-travel samples, ESBL genes were sequenced and PCR with specific primers for plasmid-encoded ß-lactamase enzymes TEM, SHV, and CTX-M group 1, 2, 8, 9, and 25 was used to confirm the presence of ESBL genes in follow-up samples. Multivariable regression analyses and mathematical modelling were used to identify predictors for acquisition and sustained carriage, and to determine household transmission rates. This study is registered with ClinicalTrials.gov, number NCT01676974. FINDINGS: 633 (34·3%) of 1847 travellers who were ESBL negative before travel and had available samples after return had acquired ESBL-E during international travel (95% CI 32·1-36·5), with the highest number of acquisitions being among those who travelled to southern Asia in 136 of 181 (75·1%, 95% CI 68·4-80·9). Important predictors for acquisition of ESBL-E were antibiotic use during travel (adjusted odds ratio 2·69, 95% CI 1·79-4·05), traveller's diarrhoea that persisted after return (2·31, 1·42-3·76), and pre-existing chronic bowel disease (2·10, 1·13-3·90). The median duration of colonisation after travel was 30 days (95% CI 29-33). 65 (11·3%) of 577 remained colonised at 12 months. CTX-M enzyme group 9 ESBLs were associated with a significantly increased risk of sustained carriage (median duration 75 days, 95% CI 48-102, p=0·0001). Onward transmission was found in 13 (7·7%) of 168 household members. The probability of transmitting ESBL-E to another household member was 12% (95% CI 5-18). INTERPRETATION: Acquisition and spread of ESBL-E during and after international travel was substantial and worrisome. Travellers to areas with a high risk of ESBL-E acquisition should be viewed as potential carriers of ESBL-E for up to 12 months after return. FUNDING: Netherlands Organisation for Health Research and Development (ZonMw).

Prolonged carriage and potential onward transmission of carbapenemase-producing Enterobacteriaceae in Dutch travelers.

AIM: The aim was to study acquisition and persistence of carbapenemase-producing Enterobacteriaceae (CPE) among travelers. MATERIALS & METHODS: Stools from 2001 travelers and 215 nontraveling household members, collected before and immediately post-travel as well as 1, 3, 6 and 12 months upon return, were screened for CPE. RESULTS: Five travelers, all visiting Asia outside the Indian subcontinent, acquired CPE. One traveler persistently carried the same OXA-244 CPE up to 6 months post-travel. Three months after travel, her co-traveling spouse also became positive for this OXA-244 CPE strain, suggesting clonal transmission within this household. CONCLUSION: Acquisition of CPE is not restricted to travelers to the Indian subcontinent and/or to travelers seeking healthcare during travel and can persist up to at least 6 months post-travel.

International travel and acquisition of multidrug-resistant Enterobacteriaceae: a systematic review.

International travel is considered to be an important risk factor for acquisition of multidrug-resistant Enterobacteriaceae (MRE). The aim of this systematic review was to determine the effect of international travel on the risk of post-travel faecal carriage of MRE. Secondary outcomes were risk factors for acquisition of MRE. A systematic search for relevant literature in seven international databases was conducted according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Articles needed to report on (i) foreign travel, (ii) screening of asymptomatic participants, (iii) antimicrobial susceptibility data and (iv) faecal Enterobacteriaceae carriage. Two researchers independently screened the abstracts, assessed the full article texts for eligibility and selected or rejected them for inclusion in the systematic review. In case of disagreement, a third researcher decided on inclusion. Eleven studies were identified. In all studies, a high prevalence (>20%) of carriage of MRE after international travel was found. The highest prevalence was observed in travellers returning from southern Asia. Foreign travel was associated with an increased risk of carriage of MRE. Further research is needed to assess if this leads to an increase in the number of infections with MRE. Systematic review registration number: PROSPERO CRD42015024973.

The Carriage Of Multiresistant Bacteria After Travel (COMBAT) prospective cohort study: methodology and design.

BACKGROUND: Antimicrobial resistance (AMR) is one of the major threats to public health around the world. Besides the intense use and misuse of antimicrobial agents as the major force behind the increase in antimicrobial resistance, the exponential increase of international travel may also substantially contribute to the emergence and spread of AMR. However, knowledge on the extent to which international travel contributes to this is still limited. The Carriage Of Multiresistant Bacteria After Travel (COMBAT) study aims to 1. determine the acquisition rate of multiresistant Enterobacteriaceae during foreign travel 2. ascertain the duration of carriage of these micro-organisms 3. determine the transmission rate within households 4. identify risk factors for acquisition, persistence of carriage and transmission of multiresistant Enterobacteriaceae. METHODS/DESIGN: The COMBAT-study is a large-scale multicenter longitudinal cohort study among travellers (n = 2001) and their non-travelling household members (n = 215). Faecal samples are collected before and immediately after travel and 1 month after return from all participants. Follow-up faecal samples are collected 3, 6 and 12 months after return from travellers (and their non-travelling household members) who acquired multiresistant Enterobacteriaceae. Questionnaires are collected from all participants at each time-point. Faecal samples are screened phenotypically for the presence of extended-spectrum beta-lactamase (ESBL) or carbapenemase-producing Enterobacteriaceae. Positive post-travel isolates from travellers with negative pre-travel samples are genotypically analysed for ESBL and carbapenemase genes with microarray and gene sequencing. DISCUSSION: The design and scale of the COMBAT-study will enable us to provide much needed detailed insights into the risks and dynamics of introduction and spread of ESBL- and carbapenemase-producing Enterobacteriaceae by healthy travellers and the potential need and measures to monitor or manage these risks. TRIAL REGISTRATION: The study is registered at clinicaltrials.gov under accession number NCT01676974.