Whole-genome Sequencing Of Isolates Research Articles

Seasonal Mortality of Wild Atlantic Menhaden (Brevoortia tyrannus) Is Caused by a Virulent Clone of Vibrio (Listonella) anguillarum; Implications for Biosecurity along the Atlantic Coastal United States

Atlantic menhaden are a highly migratory marine species in the Eastern United States that suffer from seasonal chronic mortality. Affected fish show neurologic signs referred to as spinning disease, including circling at the surface and erratic corkscrew swimming before death. We investigated three similar menhaden mortality events consistent with spinning disease in coastal New Jersey and New York between 2020 and 2021 to understand the cause. A unique strain of Vibrio (Listonella) anguillarum (serogroup O3) was detected regularly in high loads, particularly in the brains of moribund fish, by both metagenomics and bacterial isolation. The most common histopathological changes in moribund fish were hemorrhagic meningitis, encephalitis, pyknosis, and karyorrhexis of hematopoietic tissues in the kidney and spleen. Whole genome sequencing of isolates from moribund fish representing a wide spatial and temporal range showed that they were nearly identical clones, suggesting it to be a pathogenic strain circulating in the population. Though V. anguillarum is believed to be the main pathogen associated with spinning disease and mortality, Yersinia ruckeri (serotype O1) was isolated from smaller numbers of fish. Considering the highly migratory nature of Atlantic menhaden throughout the eastern United States and their use as bait for other fisheries, these findings identify potential biosecurity challenges that should be considered in Atlantic salmon aquaculture, fisheries, and emerging marine aquaculture in the region.

Novel rpsK / rpsD primer-probe assay improves detection of Campylobacter jejuni and Campylobacter coli in human stool.

Campylobacter causes bacterial enteritis, dysentery, and growth faltering in children in low- and middle-income countries (LMICs). Campylobacter spp. are fastidious organisms, and their detection often relies on culture independent diagnostic technologies, especially in LMICs. Campylobacter jejuni and Campylobacter coli are most often the infectious agents and in high income settings together account for 95% of Campylobacter infections. Several other Campylobacter species have been detected in LMIC children at an increased prevalence relative to high income settings. After doing extensive whole genome sequencing of isolates of C. jejuni and C. coli in Peru, we observed heterogeneity in the binding sites for the main species-specific PCR assay (cadF) and designed an alternative rpsKD-based qPCR assay to detect both C. jejuni and C. coli. The rpsKD-based qPCR assay identified 23% more C.jejuni/ C.coli samples than the cadF assay among 47 Campylobacter genus positive cadF negative samples verified to have C. jejuni and or C. coli with shotgun metagenomics. This assay can be expected to be useful in diagnostic studies of enteric infectious diseases and be useful in revising the attribution estimates of Campylobacter in LMICs.

Serovar-level identification of bacterial foodborne pathogens from full-length 16S rRNA gene sequencing.

The resolution of variation within species is critical for interpreting and acting on many microbial measurements. In the key foodborne pathogens Salmonella and Escherichia coli, the primary subspecies classification scheme used is serotyping: differentiating variants within these species by surface antigen profiles. Serotype prediction from whole-genome sequencing (WGS) of isolates is now seen as comparable or preferable to traditional laboratory methods where WGS is available. However, laboratory and WGS methods depend on an isolation step that is time-consuming and incompletely represents the sample when multiple strains are present. Community sequencing approaches that skip the isolation step are, therefore, of interest for pathogen surveillance. Here, we evaluated the viability of amplicon sequencing of the full-length 16S rRNA gene for serotyping Salmonella enterica and E. coli. We developed a novel algorithm for serotype prediction, implemented as an R package (Seroplacer), which takes as input full-length 16S rRNA gene sequences and outputs serovar predictions after phylogenetic placement into a reference phylogeny. We achieved over 89% accuracy in predicting Salmonella serotypes on in silico test data and identified key pathogenic serovars of Salmonella and E. coli in isolate and environmental test samples. Although serotype prediction from 16S rRNA gene sequences is not as accurate as serotype prediction from WGS of isolates, the potential to identify dangerous serovars directly from amplicon sequencing of environmental samples is intriguing for pathogen surveillance. The capabilities developed here are also broadly relevant to other applications where intraspecies variation and direct sequencing from environmental samples could be valuable.IMPORTANCEIn order to prevent and stop outbreaks of foodborne pathogens, it is important that we can detect when pathogenic bacteria are present in a food or food-associated site and identify connections between specific pathogenic bacteria present in different samples. In this work, we develop a new computational technology that allows the important foodborne pathogens Escherichia coli and Salmonella enterica to be serotyped (a subspecies level classification) from sequencing of a single-marker gene, and the 16S rRNA gene often used to surveil bacterial communities. Our results suggest current limitations to serotyping from 16S rRNA gene sequencing alone but set the stage for further progress that we consider likely given the rapid advance in the long-read sequencing technologies and genomic databases our work leverages. If this research direction succeeds, it could enable better detection of foodborne pathogens before they reach the public and speed the resolution of foodborne pathogen outbreaks.

Application of quasimetagenomics methods to define microbial diversity and subtype Listeria monocytogenes in dairy and seafood production facilities.

Microorganisms frequently colonize surfaces within food production facilities. Detection of Listeria monocytogenes in this setting relies on culture-dependent methods, but the complex dynamics of bacterial interactions within these environments and their impact on pathogen detection remain largely unexplored. To address this challenge, we applied both 16S rRNA and shotgun quasimetagenomic (enriched microbiome) sequencing of swab culture enrichments from five seafood and seven dairy production environments. Utilizing 16S rRNA amplicon sequencing, we observed variability between 355 samples taken from these 12 production facilities and a distinctive microbiome for each environment. With shotgun quasimetagenomic sequencing, we were able to assemble L. monocytogenes metagenome-assembled genomes (MAGs) from 28 of the 32 culture-positive samples. We compared these MAGs to their corresponding whole-genome sequencing assemblies, which resulted in two polyphyletic clades consisting of L. monocytogenes lineages I and II with 13,195 and 25,556 single-nucleotide polymorphism sites, respectively. The remaining four MAGs did not produce sufficient genome coverage. To understand and establish limits for pathogen detection and subtyping using shotgun quasimetagenomics, these same data sets were downsampled in slilico to produce a titration series of abundances of L. monocytogenes and analyzed. Pathogen detection was achieved for all downsampled data sets, even those with only 3× genome coverage. This study contributes to the understanding of microbial diversity within food production environments and presents insights into the level of genome coverage needed in a metagenome sequencing data set to detect, subtype, and source track a foodborne pathogenIMPORTANCEIn developed countries, the human diet is predominated by food commodities, which have been manufactured, processed, and stored in a food production facility. Little is known about the application of metagenomic sequencing approaches for detecting foodborne pathogens, such as L. monocytogenes, and characterizing microbial diversity in food production ecosystems. In this work, we investigated the utility of 16S rRNA amplicon and quasimetagenomic sequencing for the taxonomic and phylogenetic classification of Listeria culture enrichments of environmental swabs collected from dairy and seafood production facilities. We demonstrated that single-nucleotide polymorphism (SNP) analyses of L. monocytogenes metagenome-assembled genomes (MAGs) from quasimetagenomic data sets can achieve similar resolution as culture isolate whole-genome sequencing. To further understand the impact of genome coverage on MAG SNP cluster resolution, an in silico downsampling approach was employed to reduce the percentage of target pathogen sequence reads, providing an initial estimate of required MAG coverage for subtyping resolution of L. monocytogenes.

Transmission of multidrug-resistant tuberculosis in Beijing, China: An epidemiological and genomic analysis.

Understanding multidrug-resistant tuberculosis (MDR-TB) transmission patterns is crucial for controlling the disease. We aimed to identify high-risk populations and geographic settings of MDR-TB transmission. We conducted a population-based retrospective study of MDR-TB patients in Beijing from 2018 to 2020, and assessed MDR-TB recent transmission using whole-genome sequencing of isolates. Geospatial analysis was conducted with kernel density estimation. We combined TransPhylo software with epidemiological investigation data to construct transmission networks. Logistic regression analysis was utilized to identify risk factors for recent transmission. We included 241 MDR-TB patients, of which 146 (60.58%) were available for genomic analysis. Drug resistance prediction showed that resistance to fluoroquinolones (FQs) was as high as 39.74% among new cases. 36 (24.66%) of the 146 MDR strains were grouped into 12 genome clusters, suggesting recent transmission of MDR strains. 44.82% (13/29) of the clustered patients lived in the same residential community, adjacent residential community or the same street as other cases. The inferred transmission chain found a total of 6 transmission events in 3 clusters; of these, 4 transmission events occurred in residential areas and nearby public places. Logistic regression analysis revealed that being aged 25-34 years-old was a risk factor for recent transmission. The recent transmission of MDR-TB in Beijing is severe, and residential areas are common sites of transmission; high levels of FQs drug resistance suggest that FQs should be used with caution unless resistance can be ruled out by laboratory testing.

Evidence for local and international spread of Mycobacterium avium subspecies paratuberculosis through whole genome sequencing of isolates from the island of Ireland

We describe application of whole genome sequencing (WGS) to a collection of 197 Mycobacterium avium subsp paratuberculosis (MAP) isolates gathered from 122 cattle herds across 27 counties of the island of Ireland. We compare WGS to MAP diversity quantified using mycobacterial interspersed random unit – variable number tandem repeats (MIRU-VNTR). While MIRU-VNTR showed only two major types, WGS could split the 197 isolates into eight major groups. We also found six isolates corresponding to INMV 13, a novel MIRU-VNTR type for Ireland. Evidence for dispersal of MAP across Ireland via cattle movement could be discerned from the data, with mixed infections present in several herds. Furthermore, comparisons of MAP WGS data from Ireland to data from Great Britain and continental Europe revealed many instances of close genetic similarity and hence evidence for international transmission of infection. BEAST MASCOT structured coalescent analyses, with relaxed and strict molecular clocks, estimated the substitution rate to be 0.10–0.13 SNPs/site/year and disclosed greater transitions per lineage per year from Europe to Ireland, indicating transmission into Ireland. Our work therefore reveals new insight into the seeding of MAP infection across Ireland, highlighting how WGS can inform policy formulation to ultimately control MAP transmission at local, national and international scales.

Extensively Drug-Resistant Carbapenemase-Producing Pseudomonas aeruginosa and Medical Tourism from the United States to Mexico, 2018-2019.

Carbapenem-resistant Pseudomonas aeruginosa (CRPA) producing the Verona integron‒encoded metallo-β-lactamase (VIM) are highly antimicrobial drug-resistant pathogens that are uncommon in the United States. We investigated the source of VIM-CRPA among US medical tourists who underwent bariatric surgery in Tijuana, Mexico. Cases were defined as isolation of VIM-CRPA or CRPA from a patient who had an elective invasive medical procedure in Mexico during January 2018‒December 2019 and within 45 days before specimen collection. Whole-genome sequencing of isolates was performed. Thirty-eight case-patients were identified in 18 states; 31 were operated on by surgeon 1, most frequently at facility A (27/31 patients). Whole-genome sequencing identified isolates linked to surgeon 1 were closely related and distinct from isolates linked to other surgeons in Tijuana. Facility A closed in March 2019. US patients and providers should acknowledge the risk for colonization or infection after medical tourism with highly drug-resistant pathogens uncommon in the United States.

432. An Outbreak of Coronavirus Disease, 2019 (COVID-19) in a Skilled Nursing Facility – California, 2021: Description, Mitigation, Challenges, and Opportunities

BackgroundSkilled nursing facility (SNF) residents comprised 11% of all COVID-19 cases in the United States; however, they account for 43% of deaths with case fatality rates (CFR) of 26.0-33.7%. MethodsWe report an outbreak of COVID-19, from June 15 to July 21, 2020 in a 159-bed SNF with a staff of 172 that resulted in an infection rate of 97% in residents and 23% in HCWs (Figure 1). A retroactive review outlined mitigation efforts, discussed challenges, identified risk factors among residents and health care workers (HCW) for acquisition of COVID-19, and reviewed opportunities for improvement (Figure 2). Figure 1. Epi Curve of COVID-19 Outbreak in a Skilled Nursing Facility Figure 2. Timeline of COVID-19 Outbreak in a Skilled Nursing FacilityResultsFactors that contributed to the outbreak: delay in test results had an impact on cohorting; suboptimal adherence to the principles of infection prevention and control (IPC) and minimal adherence monitoring; strict criteria were used to screen for infection; the underappreciated transmissibility of COVID-19 from presymptomatic and asymptomatic persons; symptomatic HCWs who continued to work; the changing guidance on, the suboptimal use of, and an inadequate supply of personal protective equipment; poor indoor air quality due to ventilation challenges; and the important role of community/family/interfacility spread on the outbreak. Whole genome sequencing, performed in 52 samples, identified a common strain that was also found in clusters of 2 other facilities: 1 in the same geographic location, the other in a different geographic location but whose HCWs had the same zip codes as the facility (Figure 3). Certified nursing and restorative nursing assistants had the highest risk of infection with an odds ratio (OR) of 4.02 (confidence interval 1.29-12.55, p value: 0.02) when compared to registered and licensed vocational nurses. The residents’ CFR was 24%. The OR for death was increased by 10.5 (10.20-11.00) for every decade of life as was morbid obesity (BMI > 35) with an OR of 8.50. BMI as a continuous variable increased risk of mortality for every additional unit, OR 1.07 (Tables 1, 2). Whole Genome Sequencing of Isolates from a Skilled Nursing Facility Outbreak Univariate Analysis of Selected Variables Associated with Mortality among Residents at Facility A during COVID-19 Outbreak, June 19 - July 21, 2021 Multivariate Analysis of Factors Associated with Mortality from COVID-19 after Adjusting for Age among Residents (N =124) of Facility A, June 15 - July 21, 2020ConclusionWhile implementation of optimal IPC measures in the pre-COVID-19 vaccination era had no impact on the infections in residents who were likely already infected or exposed at the onset of the outbreak, these measures along with non-pharmacologic strategies were effective in halting the spread among HCWs.Disclosures All Authors: No reported disclosures

Prolonged outbreak of clonal, mupirocin-resistant methicillin-resistant Staphylococcus aureus in a neonatal intensive care unit: association with personnel and a possible environmental reservoir, analyzed using whole genome sequencing

BackgroundOutbreaks of MRSA occur in NICUs and may be difficult to control. We describe an outbreak of mupirocin-resistant MRSA, molecular epidemiology of isolates and control. MethodsMedical record review of personnel contact with infants. MRSA isolates were analyzed by whole genome sequencing (WGS); single nucleotide polymorphisms (SNPs) were identified. ResultsA 31-month outbreak of MRSA infection occurred. Weekly colonization surveillance of infants was initiated; initial prevalence was 45%. Isolates exhibited high level mupirocin-resistance. There were 3 periods of increased colonization and new infections despite implementation of multiple infection prevention interventions. During the second period, an analysis identified a frontline staff member associated with newly colonized infants whose nasal culture grew the clonal MRSA. A marked reduction in colonization followed removal from patient contact. WGS of isolates from years 1-3 showed clonality with maximum SNP differences of 33. Importantly, the year 3 isolates were more closely related to the early year 1 isolates (15-20 SNP differences) than to the late year 1 or year 2 isolates (18-33 SNP differences). Discussion/ConclusionsDuring a recrudescent MRSA outbreak due to a clonal strain, both contact with a colonized staff member and a putative environmental or personnel reservoir were associated with MRSA acquisition.

Impacts of Microbial Food Safety in China and Beyond.

Impacts of Microbial Food Safety in China and Beyond.

Tracking Salmonella enterica by whole genome sequencing of isolates recovered from broiler chickens in a poultry production system

Salmonella enterica is a major cause of foodborne diseases, and is also an important pathogenic bacterium in poultry industry. Whole genome sequencing (WGS) has become a crucial molecular typing technology used for the surveillance of the pathogenic bacteria. In the present study, we adopted WGS for tracking transmission of S. enterica in the production chain of broiler chickens. A total of 74 S. enterica strains were isolated from the different steps of breeding and slaughtering in a large production enterprise in Sichuan Province, China. The isolation rate of Salmonella was the highest in procedure of defeathering (50.0%) and evisceration (36.7%). Serotype identification showed that 74 Salmonella isolates included 7 serotypes, among which Mbandaka accounted for the highest proportions (35.1%). WGS revealed that 74 strains belonged to 7 different sequence types (STs), as well as 7 different ribosomal STs and 35 core genome STs. cgMLST-based Minimum Spanning Trees and phylogenetic tree based on the SNPs indicated that three serotypes, Mbandaka, Indiana and Kentucky, could be clonally transmitted between broiler farm and slaughterhouse. Heterogeneous resistant phenotypes and genotypes were found in two serotypes, Indiana and Kentucky. Our study indicated WGS in an accurate tool for molecular typing of S. enterica. Routine surveillance of S. enterica in the production chain of broiler chickens is needed.

Genomic epidemiology of group B streptococci spanning 10 years in an Irish maternity hospital, 2008–2017

The genomic epidemiology of group b streptococcal (GBS) isolates from the Rotunda maternity hospital, Dublin, 2008-2017, was investigated. Whole genome sequences of isolates (invasive, n = 114; non-invasive, n = 76) from infants and women were analysed using the PubMLST database (https://pubmlst.org/sagalactiae/). Serotypes III (36%), Ia (18%), V (17%), II (11%) and Ib, (9%) and sequence types (ST) 17 (23%), ST-23 (14%), ST-1 (12%) and ST-19 (7%) were most common. Core genome MLST (cgMLST) differentiated isolates of the same ST, grouped STs into five lineages congruent with known clonal complexes and identified known mother-baby pairs and suspected linked infant cases. Clonal complex (CC) 17 accounted for 40% and 22% of infant and maternal invasive cases, respectively and 21% of non-invasive isolates. CC23 and CC19 were associated with maternal disease (30%) and carriage (24%), respectively. Erythromycin (26%) and clindamycin (18%) resistance increased over the study period and was associated with presence of the erm(B) gene (55%), CC1 (33%) and CC19 (24%). A multi-resistant integrative conjugative element incorporated in the PI-1 locus was detected in CC17, an ST-12 and ST-23 isolate confirming the global dissemination of this element. All isolates possessed one or more pilus islands. Genes encoding other potential protective proteins including Sip, C5a peptidase and Srr1 were present in 100%, 99.5% and 65.8% of isolates, respectively. The srr2 gene was unique to CC17. The PubMLST.org website provides a valuable framework for genomic GBS surveillance to inform on local and global GBS epidemiology, preventive and control measures.

Population structure, case clusters, and genetic lesions associated with Canadian Salmonella 4,[5],12:i:- isolates.

Monophasic Salmonella 4,[5]:12:i:- are a major public health problem because they are one of the top five Salmonella serotypes isolated from clinical cases globally and because they can carry resistance to multiple antibiotics. A total of 811 Salmonella 4,[5]:12:i:- and S. Typhimurium whole genome sequences (WGS) were generated. The various genetic lesions causing the Salmonella 4,[5]:12:i:- genotype were identified and assessed with regards to their distribution in the population of 811 Salmonella 4,[5]:12:i:- and S. Typhimurium isolates, their geographical and temporal distribution, and their association with non-human sources. Several clades were identified in the population structure, and the largest two were associated almost exclusively with a short prophage insertion and insertion of a mobile element carrying loci encoding antibiotic and mercury resistance. IS26-mediated deletions and fljB point mutants appeared to spread clonally. 'Inconsistent' Salmonella 4,[5]:12:i:- isolates associated with specific, single amino acid changes in fljA and hin were found in a single clade composed of water, shellfish, and avian isolates. Inclusion of isolates from different case clusters identified previously by PFGE validated some of the clusters and invalidated others. Some wgMLST clusters of clinical isolates composed of very closely related isolates contained an isolate(s) with a different genetic lesion, suggesting continuing mobility of the implicated element responsible. Such cases may need to be left out of epidemiological investigations until sufficient numbers of isolates are included that statistical significance of association with sources is not impaired. Non-human sources were frequently found in or near clinical case clusters. Prospective surveillance and WGS of non-human sources and retrospective analysis by WGS of isolates from existing culture collections provides data critical for epidemiological investigations of food- and waterborne outbreaks.

Characterisation of IncI1 plasmids associated with change of phage type in isolates of Salmonella enterica serovar Typhimurium

BackgroundAcquisition of IncI1 plasmids by members of the Enterobacteriaceae sometimes leads to transfer of antimicrobial resistance and colicinogeny as well as change of phage type in Salmonella Typhimurium. Isolates of S. Typhimurium from a 2015 outbreak of food poisoning were found to contain an IncI1 plasmid implicated in change of phage type from PT135a to U307 not previously reported. The origin of the changes of phage type associated with this IncI1 plasmid was investigated. In addition, a comparison of its gene composition with that of IncI1 plasmids found in local isolates of S. Typhimurium typed as U307 from other times was undertaken. This comparison was extended to IncI1 plasmids in isolates of phage types PT6 and PT6 var. 1 which are thought to be associated with acquisition of IncI1 plasmids.ResultsAnalysis of IncI1 plasmids from whole genome sequencing of isolates implicated a gene coding for a 1273 amino acid protein present only in U307 isolates as the likely source of change of phage type. The IncI1 plasmids from PT6 and PT6 var. 1 isolates all had the ibfA gene present in IncI1 plasmid R64. This gene inhibits growth of bacteriophage BF23 and was therefore the possible source of change of phage type. A fuller comparison of the genetic composition of IncI1 plasmids from U307 isolates and PT6 and PT6 var. 1 isolates along with two IncI1 plasmids from S. Typhimurium isolates not showing change of phage type was undertaken. Plasmids were classified as either ‘Delta’ or ‘Col’ IncI1 plasmids according to whether genes between repZ and the rfsF site showed high identity to genes in the same location in R64 or ColIb-P9 plasmids respectively. Comparison of the tra gene sets and the pil gene sets across the range of sequenced plasmids identified Delta and Col plasmids with almost identical sequences for both sets of genes. This indicated a genetic recombination event leading to a switch between Delta and Col gene sets at the rfsF site. Comparisons of other gene sets showing significant variation among the sequenced plasmids are reported. Searches of the NCBI GenBank database using DNA and protein sequences of interest from the sequenced plasmids identified global IncI1 plasmids with extensive regions showing 99 to 100% identity to some of the plasmids sequenced in this study indicating evidence for widespread distribution of these plasmids.ConclusionTwo genes possibly associated with change of phage type were identified in IncI1 plasmids. IncI1 plasmids were classified as either ‘Delta’ or ‘Col’ plasmids and other sequences of significant variation among these plasmids were identified. This study offers a new perspective on the understanding of the gene composition of IncI1 plasmids. The sequences of newly sequenced IncI1 plasmids could be compared against the regions of significant sequence variation identified in this study to understand better their overall gene composition and relatedness to other IncI1 plasmids in the databases.

Dynamics of intestinal multidrug-resistant bacteria colonisation contracted by visitors to a high-endemic setting: a prospective, daily, real-time sampling study

SummaryBackgroundAntimicrobial resistance is highly prevalent in low-income and middle-income countries. International travel contributes substantially to the global spread of intestinal multidrug-resistant Gram-negative bacteria. Hundreds of millions of annual visitors to low-income and middle-income countries are all exposed to intestinal multidrug-resistant Gram-negative bacteria resulting in 30–70% of them being colonised at their return. The colonisation process in high-exposure environments is poorly documented because data have only been derived from before travel and after travel sampling. We characterised colonisation dynamics by exploring daily stool samples while visiting a low-income and middle-income countries.MethodsIn this prospective, daily, real-time sampling study 20 European visitors to Laos volunteered to provide daily stool samples and completed daily questionnaires for 22 days. Samples were initially assessed at Mahosot Hospital, Vientiane, Laos, for acquisition of extended-spectrum β-lactamase-producing (ESBL) Gram-negative bacteria followed by whole-genome sequencing of isolates at MicrobesNG, University of Birmingham, Birmingham, UK. The primary outcome of the study was to obtain data on the dynamics of intestinal multidrug-resistant bacteria acquisition.FindingsBetween Sept 18 and Sept 20, 2015, 23 volunteers were recruited, of whom 20 (87%) European volunteers were included in the final study population. Although colonisation rates were 70% at the end of the study, daily sampling revealed that all participants had acquired ESBL-producing Gram-negative bacteria at some point during the study period; the colonisation status varied day by day. Whole-genome sequencing analysis ascribed the transient pattern of colonisation to sequential acquisition of new strains, resulting in a loss of detectable colonisation by the initial multidrug-resistant Gram-negative strains. 19 (95%) participants acquired two to seven strains. Of the 83 unique strains identified (53 Escherichia coli, 10 Klebsiella spp, and 20 other ESBL-producing Gram-negative bacteria), some were shared by as many as four (20%) participants.InterpretationTo our knowledge, this is the first study to characterise in real-time the dynamics of acquiring multidrug-resistant Gram-negative bacterial colonisation during travel. Our data show multiple transient colonisation events indicative of constant microbial competition and suggest that travellers are exposed to a greater burden of multidrug-resistant bacteria than previously thought. The data emphasise the need for preventing travellers' diarrhoea and limiting antibiotic use, addressing the two major factors predisposing colonisation.FundingThe Finnish Governmental Subsidy for Health Science Research, The Scandinavian Society for Antimicrobial Chemotherapy, the Sigrid Jusélius Foundation, Biotechnology and Biological Sciences Research Council; Wellcome Trust, Medical Research Council; The Royal Society; Joint Programming Initiative on Antimicrobial Resistance, and European Research Council.

Disseminated Mycobacterium marinum in Human Immunodeficiency Virus Unmasked by Immune Reconstitution Inflammatory Syndrome.

Distinguishing disseminated Mycobacterium marinum from multifocal cutaneous disease in persons with human immunodeficiency virus/AIDS can present a diagnostic challenge, especially in the context of immune reconstitution inflammatory syndrome (IRIS). In this work, we demonstrate the utility of flow cytometry and whole genome sequencing (WGS) to diagnose disseminated M. marinum unmasked by IRIS following initiation of antiretroviral therapy. Flow cytometry demonstrated robust cytokine production by CD4 T cells in response to stimulation with M. marinum lysate. WGS of isolates from distinct lesions was consistent with clonal dissemination, supporting that preexisting disseminated M. marinum disease was uncovered by inflammatory manifestations, consistent with unmasking mycobacterial IRIS.

The study of tuberculosis outbreak in a high school—Shanghai, China, 2017–2018

To investigate the attack rate of active tuberculosis (TB) cases and detection rate of latent tuberculosis infection (LTBI) cases, and to identify possible factors contributed to the outbreak in a high school. We analyzed the investigation data of 35 teachers and 108 students, including active TB cases and their close contacts. The symptoms inquiry, tuberculin skin test (TST), and chest X-ray/CT scan were conducted in the close contact screening. We did variable-number-of-tandem-repeat genotyping and whole-genome sequencing of isolates. The time interval between the onset of symptoms and diagnosis was 36 days. The total attack rate of active TB was 7.69% (11/143), with a class attack rate of 27.78% (10/36) among students. The genetic differences between any two available isolates were less than 5 SNPs. Approximately 15.38% (22/143) of persons were LTBI cases, and the total detection rates of LTBI for students and teachers were 7.40% (8/108) and 40% (14/35), respectively. Being in the same class as the index case resulted in a significantly higher risk of being diagnosed with active TB (P = 0.0001, RR = 18.51, 95%CI (2.44,140.42)) and either LTBI and MTB infection (RR = 14.40, RR = 16.46, respectively) compared with that of students in a non-index class. The case-finding delay should be reduced to prevent the spread in classes and schools. Effective surveillance and regulated screening for student contacts should be strengthened in schools to detect infections early.

Household Transmission of Carbapenemase-producing Enterobacterales in Ontario, Canada

Data on household transmission of carbapenemase-producing Enterobacterales (CPE) remain limited. We studied risk of CPE household co-colonization and transmission in Ontario, Canada. We enrolled CPE index cases (identified via population-based surveillance from January 2015 to October 2018) and their household contacts. At months 0, 3, 6, 9, and 12, participants provided rectal and groin swabs. Swabs were cultured for CPE until September 2017, when direct polymerase chain reaction (PCR; with culture of specimens if a carbapenemase gene was detected) replaced culture. CPE risk factor data were collected by interview and combined with isolate whole-genome sequencing to determine likelihood of household transmission. Risk factors for household contact colonization were explored using a multivariable logistic regression model with generalized estimating equations. Ninety-five households with 177 household contacts participated. Sixteen (9%) household contacts in 16 (17%) households were CPE-colonized. Household transmission was confirmed in 3/177 (2%) cases, probable in 2/177 (1%), possible in 9/177 (5%), and unlikely in 2/177 (1%). Household contacts were more likely to be colonized if they were the index case's spouse (odds ratio [OR], 6.17; 95% confidence interval [CI], 1.05-36.35), if their index case remained CPE-colonized at household enrollment (OR, 7.00; 95% CI, 1.92-25.49), or if they had at least 1 set of specimens processed after direct PCR was introduced (OR, 6.46; 95% CI, 1.52-27.40). Nine percent of household contacts were CPE-colonized; 3% were a result of household transmission. Hospitals may consider admission screening for patients known to have CPE-colonized household contacts.

Strain-resolved microbiome sequencing reveals mobile elements that drive bacterial competition on a clinical timescale

BackgroundPopulations of closely related microbial strains can be simultaneously present in bacterial communities such as the human gut microbiome. We recently developed a de novo genome assembly approach that uses read cloud sequencing to provide more complete microbial genome drafts, enabling precise differentiation and tracking of strain-level dynamics across metagenomic samples. In this case study, we present a proof-of-concept using read cloud sequencing to describe bacterial strain diversity in the gut microbiome of one hematopoietic cell transplantation patient over a 2-month time course and highlight temporal strain variation of gut microbes during therapy. The treatment was accompanied by diet changes and administration of multiple immunosuppressants and antimicrobials.MethodsWe conducted short-read and read cloud metagenomic sequencing of DNA extracted from four longitudinal stool samples collected during the course of treatment of one hematopoietic cell transplantation (HCT) patient. After applying read cloud metagenomic assembly to discover strain-level sequence variants in these complex microbiome samples, we performed metatranscriptomic analysis to investigate differential expression of antibiotic resistance genes. Finally, we validated predictions from the genomic and metatranscriptomic findings through in vitro antibiotic susceptibility testing and whole genome sequencing of isolates derived from the patient stool samples.ResultsDuring the 56-day longitudinal time course that was studied, the patient’s microbiome was profoundly disrupted and eventually dominated by Bacteroides caccae. Comparative analysis of B. caccae genomes obtained using read cloud sequencing together with metagenomic RNA sequencing allowed us to identify differences in substrain populations over time. Based on this, we predicted that particular mobile element integrations likely resulted in increased antibiotic resistance, which we further supported using in vitro antibiotic susceptibility testing.ConclusionsWe find read cloud assembly to be useful in identifying key structural genomic strain variants within a metagenomic sample. These strains have fluctuating relative abundance over relatively short time periods in human microbiomes. We also find specific structural genomic variations that are associated with increased antibiotic resistance over the course of clinical treatment.

Concordance between epidemiological evaluation of probability of transmission and whole genome sequence relatedness among hospitalized patients acquiring Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae

ObjectivesWe aimed to evaluate the concordance between epidemiologically determined transmission and genetic linkage of Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae (KPC-Kp). MethodsWe included consecutive KPC-Kp carriers between December 2016 and April 2017 in a hospital endemic for KPC-Kp. We assessed epidemiological relatedness between patients by prospective investigations by the infection control team. The probability of epidemiological relatedness was classified into four groups: no suspected transmission, low, moderate and high probability of transmission. Whole-genome sequencing of isolates was performed. Genetic linkage between KPC-Kp isolates was expressed by distance between isolates in single nucleotide polymorphisms (SNPs). We established an SNP cut-off defining a different strain based on the reconstructed phylogenetic tree. We compared the epidemiological and genetic linkage of all isolates from all patients. ResultsThe study included 25 KPC-Kp carriers with 49 isolates. SNP variance was available for 1129 crossed patient-isolate pairs. Genomic linkage, based on a cut-off of 80 SNPs to define related isolates, was found in 115/708 (16.2%) of isolates with no transmission suspected epidemiologically, 27/319 (8.5%) of low, 11/26 (42.3%) of moderate and 64/76 (84.2%) of high epidemiological transmission risk determination (p < 0.001 for trend). Similar results and significant trends were shown on sensitivity analyses using a lower SNP cut-off (six SNPs) and patient-isolate unique pairs, analysing the first isolate from each patient. ConclusionsWhile significant concordance between epidemiological and genomic transmission patterns was found, epidemiological investigations of transmission are limited by the possibility of unidentified transmissions or over-estimation of associations. Genetic linkage analysis is an important aid to epidemiological transmission assessment.