scholarly journals In vitro Susceptibility of Multidrug-Resistant Pseudomonas aeruginosa following Treatment-emergent Resistance to Ceftolozane-tazobactam

2021 ◽  
Author(s):  
Abigail M. Rubio ◽  
Ellen G. Kline ◽  
Chelsea E. Jones ◽  
Liang Chen ◽  
Barry N. Kreiswirth ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Multidrug Resistant ◽  
Cross Resistance ◽  
Whole Genome ◽  
In Vitro Susceptibility ◽  
Before And After ◽  
Increased Susceptibility

We compared the in vitro susceptibility of multidrug-resistant Pseudomonas aeruginosa isolates collected before and after treatment-emergent resistance to ceftolozane-tazobactam. Median baseline and post-exposure ceftolozane-tazobactam MICs were 2 and 64 μg/mL, respectively. Whole-genome sequencing identified treatment-emergent mutations in ampC among 79% (11/14) of paired isolates. AmpC mutations were associated with cross-resistance to ceftazidime-avibactam, but increased susceptibility to piperacillin-tazobactam and imipenem. Eighty-one percent (12/16) of ceftolozane-tazobactam resistant isolates with ampC mutations were susceptible to imipenem-relebactam.

scholarly journals Ceftolozane/Tazobactam Resistance and Mechanisms in Carbapenem-Nonsusceptible Pseudomonas aeruginosa

mSphere ◽  
2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Jocelyn Qi-Min Teo ◽  
Jie Chong Lim ◽  
Cheng Yee Tang ◽  
Shannon Jing-Yi Lee ◽  
Si Hui Tan ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Molecular Epidemiology ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Antimicrobial Agents ◽  
Resistance Mechanisms ◽  
Cross Resistance ◽  
Whole Genome ◽  
Genotypic Resistance ◽  
Content Type

ABSTRACT This study established the in vitro activity of ceftolozane/tazobactam (C/T) and its genotypic resistance mechanisms by whole-genome sequencing (WGS) in 195 carbapenem-nonsusceptible Pseudomonas aeruginosa (CNSPA) clinical isolates recovered from Singapore between 2009 and 2020. C/T susceptibility rates were low, at 37.9%. Cross-resistance to ceftazidime/avibactam was observed, although susceptibility to the agent was slightly higher, at 41.0%. Whole-genome sequencing revealed that C/T resistance was largely mediated by the presence of horizontally acquired β-lactamases, especially metallo-β-lactamases. These were primarily disseminated in well-recognized high-risk clones belonging to sequence types (ST) 235, 308, and 179. C/T resistance was also observed in several non-carbapenemase-producing isolates, in which resistance was likely mediated by β-lactamases and, to a smaller extent, mutations in AmpC-related genes. There was no obvious mechanism of resistance observed in five isolates. The high C/T resistance highlights the limited utility of the agent as an empirical agent in our setting. Knowledge of local molecular epidemiology is crucial in determining the potential of therapy with novel agents. IMPORTANCE Pseudomonas aeruginosa infection is one of the most difficult health care-associated infections to treat due to the ability of the organism to acquire a multitude of resistance mechanisms and express the multidrug resistance phenotype. Ceftolozane/tazobactam (C/T), a novel β-lactam/β-lactamase inhibitor combination, addresses an unmet medical need in patients with these multidrug-resistant P. aeruginosa infections. Our findings demonstrate geographical variation in C/T susceptibility owing to the distinct local molecular epidemiology. This study adds on to the growing knowledge of C/T resistance, particularly mutational resistance, and will aid in the design of future β-lactams and β-lactamase inhibitors. WGS proved to be a useful tool to understand the P. aeruginosa resistome and its contribution to emerging resistance in novel antimicrobial agents.

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

2016 ◽  
Vol 54 (7) ◽  
pp. 1782-1788 ◽  
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.

Bronchoscope-associated clusters of multidrug-resistant Pseudomonas aeruginosa and carbapenem-resistant Klebsiella pneumoniae

2018 ◽  
Vol 40 (1) ◽  
pp. 40-46 ◽  
Author(s):  
Alison L. Galdys ◽  
Jane W. Marsh ◽  
Edgar Delgado ◽  
A. William Pasculle ◽  
Marissa Pacey ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Klebsiella Pneumoniae ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Genetic Relatedness ◽  
Multidrug Resistant ◽  
Molecular Testing ◽  
Whole Genome ◽  
Carbapenem Resistant ◽  
Clinical Culture

AbstractObjectiveRecovery of multidrug-resistant (MDR) Pseudomonas aeruginosa and Klebsiella pneumoniae from a cluster of patients in the medical intensive care unit (MICU) prompted an epidemiologic investigation for a common exposure.MethodsClinical and microbiologic data from MICU patients were retrospectively reviewed, MICU bronchoscopes underwent culturing and borescopy, and bronchoscope reprocessing procedures were reviewed. Bronchoscope and clinical MDR isolates epidemiologically linked to the cluster underwent molecular typing using pulsed-field gel electrophoresis (PFGE) followed by whole-genome sequencing.ResultsOf the 33 case patients, 23 (70%) were exposed to a common bronchoscope (B1). Both MDR P. aeruginosa and K. pneumonia were recovered from the bronchoscope’s lumen, and borescopy revealed a luminal defect. Molecular testing demonstrated genetic relatedness among case patient and B1 isolates, providing strong evidence for horizontal bacterial transmission. MDR organism (MDRO) recovery in 19 patients was ultimately linked to B1 exposure, and 10 of 19 patients were classified as belonging to an MDRO pseudo-outbreak.ConclusionsSurveillance of bronchoscope-derived clinical culture data was important for early detection of this outbreak, and whole-genome sequencing was important for the confirmation of findings. Visualization of bronchoscope lumens to confirm integrity should be a critical component of device reprocessing.

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

2017 ◽  
Vol 61 (8) ◽  
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.

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.

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