scholarly journals Generation of Tetracycline and Rifamycin Resistant Chlamydia Suis Recombinants

2021 ◽  
Vol 12 ◽  
Author(s):  
Hanna Marti ◽  
Sankhya Bommana ◽  
Timothy D. Read ◽  
Theresa Pesch ◽  
Barbara Prähauser ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Homologous Recombination ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Field Isolate ◽  
Tetracycline Resistance ◽  
Whole Genome ◽  
Gene Encoding ◽  
Obligate Intracellular

The Chlamydiaceae are a family of obligate intracellular, gram-negative bacteria known to readily exchange DNA by homologous recombination upon co-culture in vitro, allowing the transfer of antibiotic resistance residing on the chlamydial chromosome. Among all the obligate intracellular bacteria, only Chlamydia (C.) suis naturally integrated a tetracycline resistance gene into its chromosome. Therefore, in order to further investigate the readiness of Chlamydia to exchange DNA and especially antibiotic resistance, C. suis is an excellent model to advance existing co-culture protocols allowing the identification of factors crucial to promote homologous recombination in vitro. With this strategy, we co-cultured tetracycline-resistant with rifamycin group-resistant C. suis, which resulted in an allover recombination efficiency of 28%. We found that simultaneous selection is crucial to increase the number of recombinants, that sub-inhibitory concentrations of tetracycline inhibit rather than promote the selection of double-resistant recombinants, and identified a recombination-deficient C. suis field isolate, strain SWA-110 (1-28b). While tetracycline resistance was detected in field isolates, rifampicin/rifamycin resistance (RifR) had to be induced in vitro. Here, we describe the protocol with which RifR C. suis strains were generated and confirmed. Subsequent whole-genome sequencing then revealed that G530E and D461A mutations in rpoB, a gene encoding for the β-subunit of the bacterial RNA polymerase (RNAP), was likely responsible for rifampicin and rifamycin resistance, respectively. Finally, whole-genome sequencing of recombinants obtained by co-culture revealed that recombinants picked from the same plate may be sibling clones and confirmed C. suis genome plasticity by revealing variable, apparently non-specific areas of recombination.

scholarly journals The Genetic Diversification of a Single Bluetongue Virus Strain Using an In Vitro Model of Alternating-Host Transmission

Viruses ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 1038 ◽  
Author(s):  
Jennifer H. Kopanke ◽  
Justin S. Lee ◽  
Mark D. Stenglein ◽  
Christie E. Mayo
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Genetic Stability ◽  
Bluetongue Virus ◽  
Genomic Structure ◽  
Field Isolate ◽  
Viral Population ◽  
Whole Genome ◽  
Single Nucleotide Variants

Bluetongue virus (BTV) is an arbovirus that has been associated with dramatic epizootics in both wild and domestic ruminants in recent decades. As a segmented, double-stranded RNA virus, BTV can evolve via several mechanisms due to its genomic structure. However, the effect of BTV’s alternating-host transmission cycle on the virus’s genetic diversification remains poorly understood. Whole genome sequencing approaches offer a platform for investigating the effect of host-alternation across all ten segments of BTV’s genome. To understand the role of alternating hosts in BTV’s genetic diversification, a field isolate was passaged under three different conditions: (i) serial passages in Culicoides sonorensis cells, (ii) serial passages in bovine pulmonary artery endothelial cells, or (iii) alternating passages between insect and bovine cells. Aliquots of virus were sequenced, and single nucleotide variants were identified. Measures of viral population genetics were used to quantify the genetic diversification that occurred. Two consensus variants in segments 5 and 10 occurred in virus from all three conditions. While variants arose across all passages, measures of genetic diversity remained largely similar across cell culture conditions. Despite passage in a relaxed in vitro system, we found that this BTV isolate exhibited genetic stability across passages and conditions. Our findings underscore the valuable role that whole genome sequencing may play in improving understanding of viral evolution and highlight the genetic stability of BTV.

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

2020 ◽  
Vol 58 (4) ◽  
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.

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

2019 ◽  
Vol 9 (1) ◽  
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.

Advancing RNA Virus Discovery and Biology with Whole Genome Sequencing

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.

scholarly journals Integrating Culture-based Antibiotic Resistance Profiles with Whole-genome Sequencing Data for 11,087 Clinical Isolates

2019 ◽  
Vol 17 (2) ◽  
pp. 169-182 ◽  
Author(s):  
Valentina Galata ◽  
Cédric C. Laczny ◽  
Christina Backes ◽  
Georg Hemmrich-Stanisak ◽  
Susanne Schmolke ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Clinical Isolates ◽  
Whole Genome Sequencing Data ◽  
Whole Genome ◽  
Sequencing Data

scholarly journals Whole-Genome Sequencing for Detecting Antimicrobial Resistance in Nontyphoidal Salmonella

2016 ◽  
Vol 60 (9) ◽  
pp. 5515-5520 ◽  
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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