Whole-Genome Sequencing of a Brucella melitensis Strain (BMWS93) Isolated from a Bank Clerk and Exhibiting Complete Resistance to Rifampin

Human brucellosis has become the most severe public health problem in the Ulanqab region of Inner Mongolia, China. Brucella melitensis BMWS93 was obtained from a blood sample taken from a bank clerk in the Ulanqab region of Inner Mongolia, China, and antimicrobial susceptibility testing in vitro showed no zone of inhibition, which confirmed resistance to rifampin. Therefore, whole-genome sequencing of this isolate was performed to better understand the mechanism of this resistance.

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Whole Genome Sequencing for Tracing Geographical Origin of Imported Cases of Human Brucellosis in Sweden

Microorganisms ◽

10.3390/microorganisms7100398 ◽

2019 ◽

Vol 7 (10) ◽

pp. 398 ◽

Cited By ~ 4

Author(s):

Sacchini ◽

Wahab ◽

Di Giannatale ◽

Zilli ◽

Abass ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Tandem Repeats ◽

Brucella Melitensis ◽

Geographic Origin ◽

Variable Number ◽

Human Brucellosis ◽

Whole Genome ◽

Multiple Loci ◽

Imported Cases

Human infections with Brucella melitensis are occasionally reported in Sweden, despite the fact that the national flocks of sheep and goats are officially free from brucellosis. The aim of our study was to analyze 103 isolates of B. melitensis collected from patients in Sweden between 1994 and 2016 and determine their putative geographic origin using whole genome sequencing (WGS)-based tools. The majority of the strains were assigned to East Mediterranean and African lineages. Both in silico Multiple Loci VNTR (Variable Number of Tandem Repeats) Analysis (MLVA) and core genome Multilocus Sequence Typing (cgMLST) analyses identified countries of the Middle East as the most probable source of origin of the majority of the strains. Isolates collected from patients with travel history to Iraq or Syria were often associated with genotypes from Turkey, as the cgMLST profiles from these countries clustered together. Sixty strains were located within a distance of 20 core genes to related genotypes from the publicly available database, and for eighteen isolates, the closest genotype was different by more than 50 loci. Our study showed that WGS based tools are effective in tracing back the geographic origin of infection of patients with unknown travel status, provided that public sequences from the location of the source are available.

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Whole-Genome Sequencing for Detecting Antimicrobial Resistance in Nontyphoidal Salmonella

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01030-16 ◽

2016 ◽

Vol 60 (9) ◽

pp. 5515-5520 ◽

Cited By ~ 148

Author(s):

Patrick F. McDermott ◽

Gregory H. Tyson ◽

Claudine Kabera ◽

Yuansha Chen ◽

Cong Li ◽

...

Keyword(s):

Antimicrobial Resistance ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Resistance Genes ◽

The United States ◽

Whole Genome ◽

Content Type ◽

Structural Resistance ◽

Retail Meat

ABSTRACTLaboratory-basedin vitroantimicrobial susceptibility testing is the foundation for guiding anti-infective therapy and monitoring antimicrobial resistance trends. We used whole-genome sequencing (WGS) technology to identify known antimicrobial resistance determinants among strains of nontyphoidalSalmonellaand correlated these with susceptibility phenotypes to evaluate the utility of WGS for antimicrobial resistance surveillance. Six hundred fortySalmonellaof 43 different serotypes were selected from among retail meat and human clinical isolates that were tested for susceptibility to 14 antimicrobials using broth microdilution. The MIC for each drug was used to categorize isolates as susceptible or resistant based on Clinical and Laboratory Standards Institute clinical breakpoints or National Antimicrobial Resistance Monitoring System (NARMS) consensus interpretive criteria. Each isolate was subjected to whole-genome shotgun sequencing, and resistance genes were identified from assembled sequences. A total of 65 unique resistance genes, plus mutations in two structural resistance loci, were identified. There were more unique resistance genes (n =59) in the 104 human isolates than in the 536 retail meat isolates (n =36). Overall, resistance genotypes and phenotypes correlated in 99.0% of cases. Correlations approached 100% for most classes of antibiotics but were lower for aminoglycosides and beta-lactams. We report the first finding of extended-spectrum β-lactamases (ESBLs) (blaCTX-M1andblaSHV2a) in retail meat isolates ofSalmonellain the United States. Whole-genome sequencing is an effective tool for predicting antibiotic resistance in nontyphoidalSalmonella, although the use of more appropriate surveillance breakpoints and increased knowledge of new resistance alleles will further improve correlations.

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Whole-Genome Sequences of Brucella melitensis Strain QY1, Isolated from Sheep in Gansu, China

Genome Announcements ◽

10.1128/genomea.00896-17 ◽

2017 ◽

Vol 5 (35) ◽

Cited By ~ 4

Author(s):

Xiaoan Cao ◽

Zhaocai Li ◽

Zhongzi Lou ◽

Baoquan Fu ◽

Yongsheng Liu ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Human Disease ◽

Brucella Melitensis ◽

Whole Genome ◽

Negative Bacterium ◽

Genome Sequences ◽

Gram Negative ◽

Content Type ◽

Wild Mammals

ABSTRACT The facultative intracellular Gram-negative bacterium Brucella melitensis causes brucellosis in domestic and wild mammals, and it is a dominant pathogen responsible for human disease. This study reports the whole-genome sequencing of B. melitensis strain QY1, isolated from sheep suffering from abortion and arthritis in 2015 in Gansu, China.

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Frequency and Mechanisms of Spontaneous Fosfomycin Nonsusceptibility Observed upon Disk Diffusion Testing of Escherichia coli

Journal of Clinical Microbiology ◽

10.1128/jcm.01368-17 ◽

2017 ◽

Vol 56 (1) ◽

Cited By ~ 16

Author(s):

Aaron E. Lucas ◽

Ryota Ito ◽

Mustapha M. Mustapha ◽

Christi L. McElheny ◽

Roberta T. Mettus ◽

...

Keyword(s):

Escherichia Coli ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Multidrug Resistant ◽

Clinical Isolates ◽

Whole Genome ◽

Zone Of Inhibition ◽

Content Type ◽

E Coli ◽

Disk Diffusion Testing

ABSTRACTFosfomycin maintains activity against mostEscherichia coliclinical isolates, but the growth ofE. colicolonies within the zone of inhibition around the fosfomycin disk is occasionally observed upon susceptibility testing. We aimed to estimate the frequency of such nonsusceptible inner colony mutants and identify the underlying resistance mechanisms. Disk diffusion testing of fosfomycin was performed on 649 multidrug-resistantE. coliclinical isolates collected between 2011 and 2015. For those producing inner colonies inside the susceptible range, the parental strains and their representative inner colony mutants were subjected to MIC testing, whole-genome sequencing, reverse transcription-quantitative PCR (qRT-PCR), and carbohydrate utilization studies. Of the 649E. coliclinical isolates, 5 (0.8%) consistently produced nonsusceptible inner colonies. Whole-genome sequencing revealed the deletion ofuhpTencoding hexose-6-phosphate antiporter in 4 of theE. coliinner colony mutants, while the remaining mutant contained a nonsense mutation inuhpA. The expression ofuhpTwas absent in the mutant strains withuhpTdeletion and was not inducible in the strain with theuhpAmutation, unlike in its parental strain. All 5 inner colony mutants had reduced growth on minimal medium supplemented with glucose-6-phosphate. In conclusion, fosfomycin-nonsusceptible inner colony mutants can occur due to the loss of function or induction of UhpT but are rare among multidrug-resistantE. coliclinical strains. Considering that these mutants carry high biological costs, we suggest that fosfomycin susceptibility of strains that generate inner colony mutants can be interpreted on the basis of the zone of inhibition without accounting for the inner colonies.

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Application of Whole-Genome Sequencing Data for O-Specific Antigen Analysis andIn SilicoSerotyping of Pseudomonas aeruginosa Isolates

Journal of Clinical Microbiology ◽

10.1128/jcm.00349-16 ◽

2016 ◽

Vol 54 (7) ◽

pp. 1782-1788 ◽

Cited By ~ 17

Author(s):

Sandra Wingaard Thrane ◽

Véronique L. Taylor ◽

Ole Lund ◽

Joseph S. Lam ◽

Lars Jelsbak

Keyword(s):

Pseudomonas Aeruginosa ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Specific Antigen ◽

Whole Genome Sequencing Data ◽

Whole Genome ◽

Sequencing Data ◽

Chronic Infections ◽

Content Type

Accurate typing methods are required for efficient infection control. The emergence of whole-genome sequencing (WGS) technologies has enabled the development of genome-based methods applicable for routine typing and surveillance of bacterial pathogens. In this study, we developed thePseudomonas aeruginosaserotyper (PAst) program, which enabledin silicoserotyping ofP. aeruginosaisolates using WGS data. PAst has been made publically available as a web service and aptly facilitates high-throughput serotyping analysis. The program overcomes critical issues such as the loss ofin vitrotypeability often associated withP. aeruginosaisolates from chronic infections and quickly determines the serogroup of an isolate based on the sequence of the O-specific antigen (OSA) gene cluster. Here, PAst analysis of 1,649 genomes resulted in successful serogroup assignments in 99.27% of the cases. This frequency is rarely achievable by conventional serotyping methods. The limited number of nontypeable isolates found using PAst was the result of either a complete absence of OSA genes in the genomes or the artifact of genomic misassembly. With PAst,P. aeruginosaserotype data can be obtained from WGS information alone. PAst is a highly efficient alternative to conventional serotyping methods in relation to outbreak surveillance of serotype O12 and other high-risk clones, while maintaining backward compatibility to historical serotype data.

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Different Resistance Mechanisms for Cadazolid and Linezolid in Clostridium difficile Found by Whole-Genome Sequencing Analysis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00384-17 ◽

2017 ◽

Vol 61 (8) ◽

Cited By ~ 9

Author(s):

Patrick Caspers ◽

Hans H. Locher ◽

Philippe Pfaff ◽

Sarah Diggelmann ◽

Georg Rueedi ◽

...

Keyword(s):

Clostridium Difficile ◽

Ribosomal Protein ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Resistance Mechanisms ◽

Clinical Isolates ◽

Cross Resistance ◽

Whole Genome ◽

Content Type

ABSTRACT Cadazolid (CDZ) is a new antibiotic currently in clinical development for the treatment of Clostridium difficile infections. CDZ interferes with the bacterial protein synthesis machinery. The aim of the present study was to identify resistance mechanisms for CDZ and compare the results to those obtained for linezolid (LZD) in C. difficile by whole-genome sequencing (WGS) of strains generated by in vitro passages and to those obtained for LZD-resistant clinical isolates. Clones of C. difficile 630 selected with CDZ during 46 passages had a maximally 4-fold increase in CDZ MIC, while the LZD MIC for clones selected with LZD increased up to 16-fold. CDZ cross-resistance with LZD was maximally 4-fold, and no cross-resistance with other antibiotics tested was observed. Our data suggest that there are different resistance mechanisms for CDZ and LZD in C. difficile. Mutations after passages with CDZ were found in rplD (ribosomal protein L4) as well as in tra and rmt, whereas similar experiments with LZD showed mutations in rplC (ribosomal protein L3), reg, and tpr, indicating different resistance mechanisms. Although high degrees of variation between the sequenced genomes of the clinical isolates were observed, the same mutation in rplC was found in two clinical isolates with high LZD MICs. No mutations were found in the 23S rRNA genes, and attempts to isolate the cfr gene from resistant clinical isolates were unsuccessful. Analysis of 50% inhibitory concentrations (IC50s) determined in in vitro transcription/translation assays performed with C. difficile cell extracts from passaged clones correlated well with the MIC values for all antibiotics tested, indicating that the ribosomal mutations are causing the resistant phenotype.

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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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SureSelect targeted enrichment, a new cost effective method for the whole genome sequencing of Candidatus Liberibacter asiaticus

Scientific Reports ◽

10.1038/s41598-019-55144-4 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Weili Cai ◽

Schyler Nunziata ◽

John Rascoe ◽

Michael J. Stulberg

Keyword(s):

Whole Genome Sequencing ◽

Molecular Characterization ◽

Genome Sequencing ◽

Whole Genome Sequence ◽

Whole Genome ◽

Genome Coverage ◽

Metagenomic Sample ◽

Liberibacter Asiaticus

AbstractHuanglongbing (HLB) is a worldwide deadly citrus disease caused by the phloem-limited bacteria ‘Candidatus Liberibacter asiaticus’ (CLas) vectored by Asian citrus psyllids. In order to effectively manage this disease, it is crucial to understand the relationship among the bacterial isolates from different geographical locations. Whole genome sequencing approaches will provide more precise molecular characterization of the diversity among populations. Due to the lack of in vitro culture, obtaining the whole genome sequence of CLas is still a challenge, especially for medium to low titer samples. Hundreds of millions of sequencing reads are needed to get good coverage of CLas from an HLB positive citrus sample. In order to overcome this limitation, we present here a new method, Agilent SureSelect XT HS target enrichment, which can specifically enrich CLas from a metagenomic sample while greatly reducing cost and increasing whole genome coverage of the pathogen. In this study, the CLas genome was successfully sequenced with 99.3% genome coverage and over 72X sequencing coverage from low titer tissue samples (equivalent to 28.52 Cq using Li 16 S qPCR). More importantly, this method also effectively captures regions of diversity in the CLas genome, which provides precise molecular characterization of different strains.

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Tracing Melioidosis Back to the Source: Using Whole-Genome Sequencing To Investigate an Outbreak Originating from a Contaminated Domestic Water Supply

Journal of Clinical Microbiology ◽

10.1128/jcm.03453-14 ◽

2015 ◽

Vol 53 (4) ◽

pp. 1144-1148 ◽

Cited By ~ 18

Author(s):

Evan McRobb ◽

Derek S. Sarovich ◽

Erin P. Price ◽

Mirjam Kaestli ◽

Mark Mayo ◽

...

Keyword(s):

Water Supply ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Clinical Isolates ◽

Whole Genome ◽

Northern Australia ◽

Source Attribution ◽

Environmental Strain ◽

Content Type ◽

Groundwater Supply

Melioidosis, a disease of public health importance in Southeast Asia and northern Australia, is caused by the Gram-negative soil bacillusBurkholderia pseudomallei. Melioidosis is typically acquired through environmental exposure, and case clusters are rare, even in regions where the disease is endemic.B. pseudomalleiis classed as a tier 1 select agent by the Centers for Disease Control and Prevention; from a biodefense perspective, source attribution is vital in an outbreak scenario to rule out a deliberate release. Two cases of melioidosis within a 3-month period at a residence in rural northern Australia prompted an investigation to determine the source of exposure.B. pseudomalleiisolates from the property's groundwater supply matched the multilocus sequence type of the clinical isolates. Whole-genome sequencing confirmed the water supply as the probable source of infection in both cases, with the clinical isolates differing from the likely infecting environmental strain by just one single nucleotide polymorphism (SNP) each. For the first time, we report a phylogenetic analysis of genomewide insertion/deletion (indel) data, an approach conventionally viewed as problematic due to high mutation rates and homoplasy. Our whole-genome indel analysis was concordant with the SNP phylogeny, and these two combined data sets provided greater resolution and a better fit with our epidemiological chronology of events. Collectively, this investigation represents a highly accurate account of source attribution in a melioidosis outbreak and gives further insight into a frequently overlooked reservoir ofB. pseudomallei. Our methods and findings have important implications for outbreak source tracing of this bacterium and other highly recombinogenic pathogens.

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Advancing RNA Virus Discovery and Biology with Whole Genome Sequencing

10.21007/etd.cghs.2021.0551 ◽

2021 ◽

Author(s):

◽

Mariah Taylor ◽

Keyword(s):

Genetic Variation ◽

Amino Acid ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Rna Virus ◽

Animal Health ◽

Functional Domains ◽

Whole Genome ◽

Coding Region

Two RNA virus families that pose a threat to human and animal health are Hantaviridae and Coronaviridae. These RNA viruses which originate in wildlife continue and will continue to cause disease, and hence, it is critical that scientific research define the mechanisms as to how these viruses spillover and adapt to new hosts to become endemic. One gap in our ability to define these mechanisms is the lack of whole genome sequences for many of these viruses. To address this specific gap, I developed a versatile amplicon-based whole-genome sequencing (WGS) approach to identify viral genomes of hantaviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within reservoir and spillover hosts. In my research studies, I used the amplicon-based WGS approach to define the genetic plasticity of viral RNA within pathogenic and nonpathogenic hantavirus species. The standing genetic variation of Andes orthohantavirus and Prospect Hill orthohantavirus was mapped out and amino acid changes occurring outside of functional domains were identified within the nucleocapsid and glycoprotein. I observed several amino acid changes in functional domains of the RNA-dependent RNA polymerase, as well as single nucleotide polymorphisms (SNPs) within the 3’ non-coding region (NCR) of the S-segment. To identify whether virus adaptation would occur within the S- and L-segments we attempted to adapt hantaviruses in vitro in a spillover host model through passaging experiments. In early passages we identified few mutations in the M-segment with the majority being identified in the S-segment 3’ NCR and the L-segment. This work suggests that hantavirus adaptation occurs in the S- and L-segments although the effect of these mutants on pathology is yet to be determined. While sequencing laboratory isolates is easily accomplished, sequencing low concentrations of virus within the reservoir is a formidable task. I further translated our amplicon-based WGS approach into a pan-oligonucleotide amplicon-based WGS approach to sequence hantavirus vRNA and mRNA from reservoir and spillover hosts in Ukraine. This approach successfully identified a novel Puumala orthohantavirus (PUUV) strain in Ukraine and using Bayesian phylogenetics we found this strain to be associated with the PUUV Latvian lineage. Early during the SARS-CoV-2 pandemic, I applied the knowledge gained in the hantavirus WGS efforts to sequencing of SARS-CoV-2 from nasopharyngeal swabs collected in April 2020. The genetic diversity of 45 SARS-CoV-2 isolates was evaluated with the methods I developed. We identified D614G, a notable mutation known for increasing transmission, in over 90% of our isolates. Two major lineages distinguish SARS-CoV-2 variants worldwide, lineages A and B. While most of our isolates were found within B lineage, we also identified one isolate within lineage A. We performed in vitro work which confirmed A lineage isolates as having poor replication in the trachea as compared to the nasal cavity. Five of these isolates presented a unique array of mutations which were assessed in the keratin 18 human angiotensin-converting enzyme 2 (K18-hACE2) mouse model for its response immunologically and pathogenically. We identified a distinction of pathogenesis between the A and B lineages with emphysema being common amongst A lineage isolates. Additionally, we discovered a small cohort of likely SNPs that defined the late induction of eosinophils during infection. In summary, this work will further define the dynamics of genetic variation and plasticity within virus populations that cause disease outbreaks and will allow a deeper understanding of the virus-host relationship.

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