Clostridioides difficile Whole-Genome Sequencing Reveals Limited Within-Host Genetic Diversity in a Pediatric Cohort

ABSTRACT Whole-genome sequencing (WGS) is a highly sensitive method for identifying genetic relatedness and transmission of Clostridioides difficile strains. Previous studies suggest that as few as 3 core genome single-nucleotide variants (SNVs) discriminate between genetically distinct isolates. Because a single C. difficile colony is selected from culture for WGS, significant within-host genetic diversity could preclude identification of transmission events. To evaluate the likelihood of missed transmission events using WGS of single colonies from culture, we examined within-host genetic diversity among C. difficile isolates collected from children. We performed WGS using an Illumina MiSeq instrument on 8 C. difficile colonies randomly selected from each culture performed on stool collected from 10 children (8 children diagnosed with C. difficile infection and 2 children with asymptomatic carriage); 77/80 (96%) isolate sequences were successfully assembled. Among 8/10 (80%) children, all isolates were the same sequence type (ST). The other 2 children each had mixed infection with two STs, although one ST predominated. Among 9/10 (90%) children, isotypic isolates differed by ≤2 SNVs; an isotypic isolate in the remaining child differed by 3 to SNVs relative to the other isolates from that child. Overall, among the 77 isolates collected from 10 stool cultures, 74/77 (96%) were clonal (i.e., same ST and ≤2 core genome SNVs) to other isolates in stool culture. In summary, we identified rare C. difficile within-host genetic diversity in children, suggesting that WGS of a single colony from stool is likely to appropriately characterize isolate clonality and putative transmission events in the majority of cases.

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Comparative Whole-Genome Phylogeny of Animal, Environmental, and Human Strains Confirms the Genogroup Organization and Diversity of the Stenotrophomonas maltophilia Complex

Applied and Environmental Microbiology ◽

10.1128/aem.02919-19 ◽

2020 ◽

Vol 86 (10) ◽

Author(s):

Mélanie Mercier-Darty ◽

Guilhem Royer ◽

Brigitte Lamy ◽

Chadly Charron ◽

Olivier Lemenand ◽

...

Keyword(s):

Genetic Diversity ◽

Antimicrobial Resistance ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Resistance Genes ◽

Stenotrophomonas Maltophilia ◽

Whole Genome ◽

Content Type ◽

Genetic Organization ◽

Animal Strains

ABSTRACT The Stenotrophomonas maltophilia complex (Smc) comprises opportunistic environmental Gram-negative bacilli responsible for a variety of infections in both humans and animals. Beyond its large genetic diversity, its genetic organization in genogroups was recently confirmed through the whole-genome sequencing of human and environmental strains. As they are poorly represented in these analyses, we sequenced the whole genomes of 93 animal strains to determine their genetic background and characteristics. Combining these data with 81 newly sequenced human strains and the genomes available from RefSeq, we performed a genomic analysis that included 375 nonduplicated genomes with various origins (animal, 104; human, 226; environment, 30; unknown, 15). Phylogenetic analysis and clustering based on genome-wide average nucleotide identity confirmed and specified the genetic organization of Smc in at least 20 genogroups. Two new genogroups were identified, and two previously described groups were further divided into two subgroups each. Comparing the strains isolated from different host types and their genogroup affiliation, we observed a clear disequilibrium in certain groups. Surprisingly, some antimicrobial resistance genes, integrons, and/or clusters of attC sites lacking integron-integrase (CALIN) sequences targeting antimicrobial compounds extensively used in animals were mainly identified in animal strains. We also identified genes commonly found in animal strains coding for efflux systems. The result of a large whole-genome analysis performed by us supports the hypothesis of the putative contribution of animals as a reservoir of Stenotrophomonas maltophilia complex strains and/or resistance genes for strains in humans. IMPORTANCE Given its naturally large antimicrobial resistance profile, the Stenotrophomonas maltophilia complex (Smc) is a set of emerging pathogens of immunosuppressed and cystic fibrosis patients. As it is group of environmental microorganisms, this adaptation to humans is an opportunity to understand the genetic and metabolic selective mechanisms involved in this process. The previously reported genomic organization was incomplete, as data from animal strains were underrepresented. We added the missing piece of the puzzle with whole-genome sequencing of 93 strains of animal origin. Beyond describing the phylogenetic organization, we confirmed the genetic diversity of the Smc, which could not be estimated through routine phenotype- or matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF)-based laboratory tests. Animals strains seem to play a key role in the diversity of Smc and could act as a reservoir for mobile resistance genes. Some genogroups seem to be associated with particular hosts; the genetic support of this association and the role of the determinants/corresponding genes need to be explored.

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Streptococcus gallolyticus subsp. gallolyticus from Human and Animal Origins: Genetic Diversity, Antimicrobial Susceptibility, and Characterization of a Vancomycin-Resistant Calf Isolate Carrying avanA-Tn1546-Like Element

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04083-14 ◽

2015 ◽

Vol 59 (4) ◽

pp. 2006-2015 ◽

Cited By ~ 12

Author(s):

Beatriz Romero-Hernández ◽

Ana P. Tedim ◽

José Francisco Sánchez-Herrero ◽

Pablo Librado ◽

Julio Rozas ◽

...

Keyword(s):

Genetic Diversity ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Antibiotic Susceptibility ◽

Resistant Isolate ◽

Comparative Genomic ◽

Gene Gain ◽

Whole Genome ◽

Content Type ◽

Streptococcus Gallolyticus

ABSTRACTThe aim of this work was to characterize the antibiotic susceptibility and genetic diversity of 41Streptococcus gallolyticussubsp.gallolyticusisolates: 18 isolates obtained from animals and 23 human clinical isolates. Antibiotic susceptibility was determined by the semiautomatic Wider system and genetic diversity by pulsed-field gel electrophoresis (PFGE) with SmaI. Animal isolates grouped separately in the PFGE analysis, but no statistical differences in antimicrobial resistance were found between the two groups. The LMG 17956 sequence type 28 (ST28) strain recovered from the feces of a calf exhibited high levels of resistance to vancomycin and teicoplanin (MIC, ≥256 mg/liter). Its glycopeptide resistance mechanism was characterized by Southern blot hybridization and a primer-walking strategy, and finally its genome, determined by whole-genome sequencing, was compared with four closely relatedS. gallolyticussubsp.gallolyticusgenomes. Hybridization experiments demonstrated that a Tn1546-like element was integrated into the bacterial chromosome. In agreement with this finding, whole-genome sequencing confirmed a partial deletion of thevanY-vanZregion and partial duplication of thevanHgene. The comparative genomic analyses revealed that the LMG 17956 ST28 strain had acquired an unusually high number of transposable elements and had experienced extensive chromosomal rearrangements, as well as gene gain and loss events. In conclusion,S. gallolyticussubsp.gallolyticusisolates from animals seem to belong to lineages separate from those infecting humans. In addition, we report a glycopeptide-resistant isolate from a calf carrying a Tn1546-like element integrated into its chromosome.

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Ceftriaxone-Resistant Neisseria gonorrhoeae Isolates (2010 to 2014) in France Characterized by Using Whole-Genome Sequencing

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01568-16 ◽

2016 ◽

Vol 60 (11) ◽

pp. 6962-6964 ◽

Cited By ~ 15

Author(s):

Claire de Curraize ◽

Sylvain Kumanski ◽

Maïté Micaëlo ◽

Nelly Fournet ◽

Guy La Ruche ◽

...

Keyword(s):

Neisseria Gonorrhoeae ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Multidrug Resistant ◽

Sequence Type ◽

The Other ◽

Resistant Isolate ◽

Whole Genome ◽

Content Type ◽

Cephalosporin Resistance

ABSTRACTTwo extended-spectrum cephalosporin-resistantNeisseria gonorrhoeaeisolates were discovered among 6,340 (0.03%) French isolates between 2010 and 2014. One isolate corresponded to the F89 multidrug-resistantN. gonorrhoeaeisolate harboring apenAmosaic; whole-genome sequencing highlighted an additional R251H substitution in theftsXgene recently involved in cephalosporin resistance. The other, ceftriaxone-resistant isolate (MIC, 0.25 mg/liter) harbored the PBP2 pattern XXXVI plus a P551S substitution and belonged to sequence type ST1579 (multilocus sequence typing [MLST]).

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Evaluation of whole-genome sequencing-based subtyping methods for the surveillance of Shigella spp. and the confounding effect of mobile genetic elements in long-term outbreaks

Microbial Genomics ◽

10.1099/mgen.0.000672 ◽

2021 ◽

Vol 7 (11) ◽

Author(s):

Isabelle Bernaquez ◽

Christiane Gaudreau ◽

Pierre A. Pilon ◽

Sadjia Bekal

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Type Species ◽

Core Genome ◽

Outbreak Detection ◽

Whole Genome ◽

Content Type ◽

Link Type ◽

Interpretation Criteria

Many public health laboratories across the world have implemented whole-genome sequencing (WGS) for the surveillance and outbreak detection of foodborne pathogens. PulseNet-affiliated laboratories have determined that most single-strain foodborne outbreaks are contained within 0–10 multi-locus sequence typing (MLST)-based allele differences and/or core genome single-nucleotide variants (SNVs). In addition to being a food- and travel-associated outbreak pathogen, most Shigella spp. cases occur through continuous person-to-person transmission, predominantly involving men who have sex with men (MSM), leading to long-term and recurrent outbreaks. Continuous transmission patterns coupled to genetic evolution under antibiotic treatment pressure require an assessment of existing WGS-based subtyping methods and interpretation criteria for cluster inclusion/exclusion. An evaluation of 4 WGS-based subtyping methods [SNVPhyl, coreMLST, core genome MLST (cgMLST) and whole-genome MLST (wgMLST)] was performed on 9 foodborne-, travel- and MSM-related retrospective outbreaks from a collection of 91 Shigella flexneri and 232 Shigella sonnei isolates to determine the methods’ epidemiological concordance, discriminatory power, robustness and ability to generate stable interpretation criteria. The discriminatory powers were ranked as follows: coreMLST<SNVPhyl<cgMLST<wgMLST (range: 0.970–1.000). The genetic differences observed for non-MSM-related Shigella spp. outbreaks respect the standard 0–10 allele/SNV guideline; however, mobile genetic element (MGE)-encoded loci caused inflated genetic variation and discrepant phylogenies for prolonged MSM-related S. sonnei outbreaks via wgMLST. The S. sonnei correlation coefficients of wgMLST were also the lowest at 0.680, 0.703 and 0.712 for SNVPhyl, coreMLST and cgMLST, respectively. Plasmid maintenance, mobilization and conjugation-associated genes were found to be the main source of genetic distance inflation in addition to prophage-related genes. Duplicated alleles arising from the repeated nature of IS elements were also responsible for many false cg/wgMLST differences. The coreMLST approach was shown to be the most robust, followed by SNVPhyl and wgMLST for inter-laboratory comparability. Our results highlight the need for validating species-specific subtyping methods based on microbial genome plasticity and outbreak dynamics in addition to the importance of filtering confounding MGEs for cluster detection.

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Emergence and Within-Host Genetic Evolution of Methicillin-ResistantStaphylococcus aureusResistant to Linezolid in a Cystic Fibrosis Patient

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00720-18 ◽

2018 ◽

Vol 62 (12) ◽

Cited By ~ 7

Author(s):

Caroline Rouard ◽

Fabien Garnier ◽

Jeremy Leraut ◽

Margaux Lepainteur ◽

Lalaina Rahajamananav ◽

...

Keyword(s):

Cystic Fibrosis ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Generation Time ◽

Strong Increase ◽

Whole Genome ◽

Content Type ◽

Mrsa Infection ◽

Host Genetic ◽

Mrsa Strains

ABSTRACTMethicillin-resistantStaphylococcus aureus(MRSA) infection has increased in recent years among cystic fibrosis (CF) patients. Linezolid (LZD) is one of the antistaphylococcal antibiotics widely used in this context. Although LZD resistance is rare, it has been described as often associated with long-term treatments. Thirteen MRSA strains isolated over 5 years from one CF patient were studied for LZD resistance emergence and subjected to whole-genome sequencing (WGS). Resistance emerged after three 15-day LZD therapeutic regimens over 4 months. It was associated with the mutation of G to T at position 2576 (G2576T) in all 5rrlcopies, along with a very high MIC (>256 mg/liter) and a strong increase in the generation time. Resistant strains isolated during the ensuing LZD therapeutic regimens and until 13 months after LZD stopped harbored only 3 or 4 mutatedrrlcopies, associated with lower MICs (8 to 32 mg/liter) and low to moderate generation time increases. Despite these differences, whole-genome sequencing allowed us to determine that all isolates, including the susceptible one isolated before LZD treatment, belonged to the same lineage. In conclusion, LZD resistance can emerge rapidly in CF patients and persist without linezolid selective pressure in colonizing MRSA strains belonging to the same lineage.

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Mycobacterium chimaera colonisation of heater–cooler units (HCU) in Western Australia, 2015: investigation of possible iatrogenic infection using whole genome sequencing

Eurosurveillance ◽

10.2807/1560-7917.es.2016.21.46.30396 ◽

2016 ◽

Vol 21 (46) ◽

Cited By ~ 14

Author(s):

James Owen Robinson ◽

Geoffrey Wallace Coombs ◽

David John Speers ◽

Terillee Keehner ◽

Anthony David Keil ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Western Australia ◽

Genetic Relatedness ◽

The Other ◽

Whole Genome ◽

Pleural Biopsy

Following the reported link between heater–cooler unit (HCU) colonisation with Mycobacterium chimaera and endocarditis, mycobacterial sampling of all HCUs in use in Western Australia was initiated from August 2015, revealing M. chimaera colonisation in 10 of 15 HCUs. After M. chimaera was isolated from a pleural biopsy from a cardiothoracic patient who may have been exposed to a colonised HCU, a whole genome sequencing investigation was performed involving 65 specimens from 15 HCUs across five hospitals to assess if this infection was related to the HCU. Genetic relatedness was found between the 10 HCU M. chimaera isolates from four hospitals. However the M. chimaera isolate from the cardiothoracic patient was not genetically related to the HCU M. chimaera isolates from that hospital, nor to the other HCU isolates, indicating that the HCUs were not the source of the infection in this patient.

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Whole-Genome Sequencing of Recent Listeria monocytogenes Isolates from Germany Reveals Population Structure and Disease Clusters

Journal of Clinical Microbiology ◽

10.1128/jcm.00119-18 ◽

2018 ◽

Vol 56 (6) ◽

Cited By ~ 20

Author(s):

Sven Halbedel ◽

Rita Prager ◽

Stephan Fuchs ◽

Eva Trost ◽

Guido Werner ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Core Genome ◽

Cluster Detection ◽

Whole Genome ◽

Single Nucleotide ◽

Content Type ◽

Disease Clusters ◽

Foodborne Outbreaks

ABSTRACT Listeria monocytogenes causes foodborne outbreaks with high mortality. For improvement of outbreak cluster detection, the German consiliary laboratory for listeriosis implemented whole-genome sequencing (WGS) in 2015. A total of 424 human L. monocytogenes isolates collected in 2007 to 2017 were subjected to WGS and core-genome multilocus sequence typing (cgMLST). cgMLST grouped the isolates into 38 complexes, reflecting 4 known and 34 unknown disease clusters. Most of these complexes were confirmed by single nucleotide polymorphism (SNP) calling, but some were further differentiated. Interestingly, several cgMLST cluster types were further subtyped by pulsed-field gel electrophoresis, partly due to phage insertions in the accessory genome. Our results highlight the usefulness of cgMLST for routine cluster detection but also show that cgMLST complexes require validation by methods providing higher typing resolution. Twelve cgMLST clusters included recent cases, suggesting activity of the source. Therefore, the cgMLST nomenclature data presented here may support future public health actions.

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Characterization of HypermutatorPseudomonas aeruginosaIsolates from Patients with Cystic Fibrosis in Australia

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02538-18 ◽

2019 ◽

Vol 63 (4) ◽

Cited By ~ 5

Author(s):

Vanessa E. Rees ◽

Deanna S. Deveson Lucas ◽

Carla López-Causapé ◽

Yuling Huang ◽

Tom Kotsimbos ◽

...

Keyword(s):

Genetic Diversity ◽

Health Care ◽

Cystic Fibrosis ◽

Core Genome ◽

Genetic Relatedness ◽

Phylogenetic Analyses ◽

Whole Genome ◽

Genome Sequences ◽

Content Type

ABSTRACTHypermutablePseudomonas aeruginosaisolates (hypermutators) have been identified in patients with cystic fibrosis (CF) and are associated with reduced lung function. Hypermutators display a greatly increased mutation rate and an enhanced ability to become resistant to antibiotics during treatment. Their prevalence has been established among patients with CF, but it has not been determined for patients with CF in Australia. This study aimed to determine the prevalence of hypermutableP. aeruginosaisolates from adult patients with CF from a health care institution in Australia and to characterize the genetic diversity and antibiotic susceptibility of these isolates. A total of 59 P. aeruginosaclinical isolates from patients with CF were characterized. For all isolates, rifampin (RIF) mutation frequencies and susceptibility to a range of antibiotics were determined. Of the 59 isolates, 13 (22%) were hypermutable. Whole-genome sequences were determined for all hypermutable isolates. Core genome polymorphisms were used to assess genetic relatedness of the isolates, both to each other and to a sample of previously characterizedP. aeruginosastrains. Phylogenetic analyses showed that the hypermutators were from divergent lineages and that hypermutator phenotype was mostly the result of mutations inmutLor, less commonly, inmutS. Hypermutable isolates also contained a range of mutations that are likely associated with adaptation ofP. aeruginosato the CF lung environment. Multidrug resistance was more prevalent in hypermutable than nonhypermutable isolates (38% versus 22%). This study revealed that hypermutableP. aeruginosastrains are common among isolates from patients with CF in Australia and are implicated in the emergence of antibiotic resistance.

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Methodology for Whole-Genome Sequencing of Methicillin-Resistant Staphylococcus aureus Isolates in a Routine Hospital Microbiology Laboratory

Journal of Clinical Microbiology ◽

10.1128/jcm.00180-19 ◽

2019 ◽

Vol 57 (6) ◽

Cited By ~ 9

Author(s):

Kathy E. Raven ◽

Beth Blane ◽

Danielle Leek ◽

Carol Churcher ◽

Paula Kokko-Gonzales ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Genetic Relatedness ◽

Turnaround Time ◽

Whole Genome ◽

Test Panel ◽

Methicillin Resistant ◽

Content Type ◽

Outbreak Investigations

ABSTRACT There is growing evidence for the value of bacterial whole-genome sequencing in hospital outbreak investigations. Our aim was to develop methods that support efficient and accurate low-throughput clinical sequencing of methicillin-resistant Staphylococcus aureus (MRSA) isolates. Using a test panel of 25 MRSA isolates previously associated with outbreak investigations, we devised modifications to library preparation that reduced the processing time by 1 hour. We determined the maximum number of isolates that could be sequenced per run using an Illumina MiniSeq platform and a 13-hour (overnight) run time, which equated to 21 MRSA isolates and 3 controls (no template, positive, and negative). Repeatability and reproducibility assays based on this sequencing methodology demonstrated 100% accuracy in assigning species and sequence type (ST) and in detecting mecA. Established genetic relatedness between isolates was recapitulated. Quality control (QC) metrics were evaluated over nine sequencing runs. Of the test panel MRSA genomes, 168/173 (97%) passed QC metrics based on the correct species assigned, detection of mecA and ST, and depth/coverage metrics. An evaluation of contamination in these 9 runs showed that positive and negative controls and test MRSA sequence files contained <0.14% and <0.48% of fragments that matched another species, respectively. Deliberate contamination experiments confirmed that this was insufficient to impact data interpretation. These methods support reliable and reproducible clinical MRSA sequencing with a turnaround time (from DNA extraction to availability of data files) of 24 hours.

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Expanding U.S. Laboratory Capacity for Neisseria gonorrhoeae Antimicrobial Susceptibility Testing and Whole-Genome Sequencing through the CDC's Antibiotic Resistance Laboratory Network

Journal of Clinical Microbiology ◽

10.1128/jcm.01461-19 ◽

2020 ◽

Vol 58 (4) ◽

Cited By ~ 2

Author(s):

Ellen N. Kersh ◽

Cau D. Pham ◽

John R. Papp ◽

Robert Myers ◽

Richard Steece ◽

...

Keyword(s):

Public Health ◽

Antibiotic Resistance ◽

Neisseria Gonorrhoeae ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Antimicrobial Susceptibility ◽

Susceptibility Testing ◽

Whole Genome ◽

Content Type ◽

Laboratory Capacity

ABSTRACT U.S. gonorrhea rates are rising, and antibiotic-resistant Neisseria gonorrhoeae (AR-Ng) is an urgent public health threat. Since implementation of nucleic acid amplification tests for N. gonorrhoeae identification, the capacity for culturing N. gonorrhoeae in the United States has declined, along with the ability to perform culture-based antimicrobial susceptibility testing (AST). Yet AST is critical for detecting and monitoring AR-Ng. In 2016, the CDC established the Antibiotic Resistance Laboratory Network (AR Lab Network) to shore up the national capacity for detecting several resistance threats including N. gonorrhoeae. AR-Ng testing, a subactivity of the CDC’s AR Lab Network, is performed in a tiered network of approximately 35 local laboratories, four regional laboratories (state public health laboratories in Maryland, Tennessee, Texas, and Washington), and the CDC’s national reference laboratory. Local laboratories receive specimens from approximately 60 clinics associated with the Gonococcal Isolate Surveillance Project (GISP), enhanced GISP (eGISP), and the program Strengthening the U.S. Response to Resistant Gonorrhea (SURRG). They isolate and ship up to 20,000 isolates to regional laboratories for culture-based agar dilution AST with seven antibiotics and for whole-genome sequencing of up to 5,000 isolates. The CDC further examines concerning isolates and monitors genetic AR markers. During 2017 and 2018, the network tested 8,214 and 8,628 N. gonorrhoeae isolates, respectively, and the CDC received 531 and 646 concerning isolates and 605 and 3,159 sequences, respectively. In summary, the AR Lab Network supported the laboratory capacity for N. gonorrhoeae AST and associated genetic marker detection, expanding preexisting notification and analysis systems for resistance detection. Continued, robust AST and genomic capacity can help inform national public health monitoring and intervention.

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