Lipid A-Ara4N as an alternate pathway for (colistin) resistance in Klebsiella pneumonia isolates in Pakistan

Abstract Objectives This study aimed to explore mechanism of colistin resistance amongst Klebsiella pneumoniae isolates through plasmid mediated mcr-1 gene in Pakistan. Carbapenem and Colistin resistant K. pneumoniae isolates (n = 34) stored at − 80 °C as part of the Aga Khan University Clinical Laboratory strain bank were randomly selected and subjected to mcr-1 gene PCR. To investigate mechanisms of resistance, other than plasmid mediated mcr-1 gene, whole genome sequencing was performed on 8 clinical isolates, including 6 with colistin resistance (MIC > 4 μg/ml) and 2 with intermediate resistance to colistin (MIC > 2 μg/ml). Results RT-PCR conducted revealed absence of mcr-1 gene in all isolates tested. Whole genome sequencing results revealed modifications in Lipid A-Ara4N pathway. Modifications in Lipid A-Ara4N pathway were detected in ArnA_ DH/FT, UgdH, ArnC and ArnT genes. Mutation in ArnA_ DH/FT gene were detected in S3, S5, S6 and S7 isolates. UgdH gene modifications were found in all isolates except S3, mutations in ArnC were present in all except S1, S2 and S8 and ArnT were detected in all except S4 and S7. In the absence of known mutations linked with colistin resistance, lipid pathway modifications may possibly explain the phenotype resistance to colistin, but this needs further exploration.

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Whole Genome Sequencing in the Clinical Laboratory

Modern Clinical Molecular Techniques ◽

10.1007/978-1-4614-2170-2_5 ◽

2012 ◽

pp. 67-78

Author(s):

Tina Hambuch ◽

Brad Sickler ◽

Arnold Liao ◽

Suneer Jain ◽

Philip D. Cotter

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Clinical Laboratory ◽

Whole Genome

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False COVID-19 cases due to contamination by inactivated virus vaccine

Clinical Infectious Diseases ◽

10.1093/cid/ciab684 ◽

2021 ◽

Author(s):

Kelvin Kai-Wang To ◽

Xin Li ◽

David Christopher Lung ◽

Jonathan Daniel Ip ◽

Wan-Mui Chan ◽

...

Keyword(s):

Public Health ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Virus Vaccine ◽

False Positive ◽

Spike Protein ◽

Whole Genome ◽

Rt Pcr ◽

Health Measures ◽

Respiratory Specimens

Abstract A false-positive SARS-CoV-2 RT-PCR result can lead to unnecessary public-health measures. We report two individuals whose respiratory specimens were contaminated by inactivated SARS-CoV-2 vaccine strain(CoronaVac), likely at vaccination premises. Incidentally, whole-genome sequencing of CoronaVac showed adaptive deletions on the spike protein, which do not result in observable changes of antigenicity.

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Precision medicine integrating whole-genome sequencing, comprehensive metabolomics, and advanced imaging

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1909378117 ◽

2020 ◽

Vol 117 (6) ◽

pp. 3053-3062 ◽

Cited By ~ 9

Author(s):

Ying-Chen Claire Hou ◽

Hung-Chun Yu ◽

Rick Martin ◽

Elizabeth T. Cirulli ◽

Natalie M. Schenker-Ahmed ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Precision Medicine ◽

Genome Sequencing ◽

Clinical Laboratory ◽

Descriptive Analysis ◽

Disease Diagnosis ◽

Whole Genome ◽

Advanced Imaging ◽

Family Medical History ◽

Deep Phenotyping

Genome sequencing has established clinical utility for rare disease diagnosis. While increasing numbers of individuals have undergone elective genome sequencing, a comprehensive study surveying genome-wide disease-associated genes in adults with deep phenotyping has not been reported. Here we report the results of a 3-y precision medicine study with a goal to integrate whole-genome sequencing with deep phenotyping. A cohort of 1,190 adult participants (402 female [33.8%]; mean age, 54 y [range 20 to 89+]; 70.6% European) had whole-genome sequencing, and were deeply phenotyped using metabolomics, advanced imaging, and clinical laboratory tests in addition to family/medical history. Of 1,190 adults, 206 (17.3%) had at least 1 genetic variant with pathogenic (P) or likely pathogenic (LP) assessment that suggests a predisposition of genetic risk. A multidisciplinary clinical team reviewed all reportable findings for the assessment of genotype and phenotype associations, and 137 (11.5%) had genotype and phenotype associations. A high percentage of genotype and phenotype associations (>75%) was observed for dyslipidemia (n = 24), cardiomyopathy, arrhythmia, and other cardiac diseases (n = 42), and diabetes and endocrine diseases (n = 17). A lack of genotype and phenotype associations, a potential burden for patient care, was observed in 69 (5.8%) individuals with P/LP variants. Genomics and metabolomics associations identified 61 (5.1%) heterozygotes with phenotype manifestations affecting serum metabolite levels in amino acid, lipid and cofactor, and vitamin pathways. Our descriptive analysis provides results on the integration of whole-genome sequencing and deep phenotyping for clinical assessments in adults.

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Complete Genome Sequence of the Gut Commensal and Laboratory Strain Enterococcus faecium 64/3

Genome Announcements ◽

10.1128/genomea.01275-15 ◽

2015 ◽

Vol 3 (6) ◽

Cited By ~ 5

Author(s):

Jennifer K. Bender ◽

Stefan Fiedler ◽

Ingo Klare ◽

Guido Werner

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Genome Sequence ◽

Complete Genome Sequence ◽

Enterococcus Faecium ◽

Complete Genome ◽

Laboratory Strain ◽

Whole Genome ◽

Coding Sequences

The genome sequence of the commensal and widely used laboratory strain Enterococcus faecium 64/3 was resolved by means of PacificBioscience and Illumina whole-genome sequencing. The genome comprises 2,575,333 bp with 2,382 coding sequences as assigned by NCBI.

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Detection of SARS-CoV-2 from Patient Fecal Samples by Whole Genome Sequencing

10.21203/rs.3.rs-48863/v1 ◽

2020 ◽

Author(s):

Andreas Papoutsis ◽

Thomas Borody ◽

Siba Dolai ◽

Jordan Daniels ◽

Skylar Steinberg ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Genome Analysis ◽

Nasopharyngeal Swab ◽

Whole Genome ◽

Rt Pcr ◽

Whole Genome Analysis ◽

Fecal Samples ◽

Oral Transmission ◽

Study Participants

Abstract Background SARS-CoV-2 has been detected not only in respiratory secretions, but also in stool collections. Here were sought to identify SARS-CoV-2 by enrichment NGS from fecal samples, and to utilize whole genome analysis to characterize SARS-CoV-2 mutational variations in COVID-19 patients. Results Study participants underwent testing for SARS-CoV-2 from fecal samples by whole genome enrichment NGS (n = 14), and RT-PCR nasopharyngeal swab analysis (n = 12). The concordance of SARS-CoV-2 detection by enrichment NGS from stools with RT-PCR nasopharyngeal analysis was 100%. Unique variants were identified in four patients, with a total of 33 different mutations among those in which SARS-CoV-2 was detected by whole genome enrichment NGS. Conclusion These results highlight the potential viability of SARS-CoV-2 in feces, its ongoing mutational accumulation, and its possible role in fecal-oral transmission. This study also elucidates the advantages of SARS-CoV-2 enrichment NGS, which may be a key methodology to document complete viral eradication.

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A Retrospective Whole-Genome Sequencing Analysis of Carbapenem and Colistin-Resistant Klebsiella pneumoniae Nosocomial Strains Isolated during an MDR Surveillance Program

Antibiotics ◽

10.3390/antibiotics9050246 ◽

2020 ◽

Vol 9 (5) ◽

pp. 246 ◽

Cited By ~ 1

Author(s):

Bernardina Gentile ◽

Antonella Grottola ◽

Gabriella Orlando ◽

Giulia Fregni Serpini ◽

Claudia Venturelli ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Capsular Polysaccharide ◽

University Hospital ◽

Surveillance Program ◽

Whole Genome ◽

Sequencing Analysis ◽

Nucleotide Polymorphisms ◽

Colistin Resistance

Multidrug-resistant Klebsiella pneumoniae (MDR Kp), in particular carbapenem-resistant Kp (CR-Kp), has become endemic in Italy, where alarming data have been reported on the spread of colistin-resistant CR-Kp (CRCR-Kp). During the period 2013–2014, 27 CRCR-Kp nosocomial strains were isolated within the Modena University Hospital Policlinico (MUHP) multidrug resistance surveillance program. We retrospectively investigated these isolates by whole-genome sequencing (WGS) analysis of the resistome, virulome, plasmid content, and core single nucleotide polymorphisms (cSNPs) in order to gain insights into their molecular epidemiology. The in silico WGS analysis of the resistome revealed the presence of genes, such as blaKPC, related to the phenotypically detected resistances to carbapenems. Concerning colistin resistance, the plasmidic genes mcr 1–9 were not detected, while known and new genetic variations in mgrB, phoQ, and pmrB were found. The virulome profile revealed the presence of type-3 fimbriae, capsular polysaccharide, and iron acquisition system genes. The detected plasmid replicons were classified as IncFIB(pQil), IncFIB(K), ColRNAI, IncX3, and IncFII(K) types. The cSNPs genotyping was consistent with the multi locus sequence typing (MLST) and with the distribution of mutations related to colistin resistance genes. In a nosocomial drug resistance surveillance program, WGS proved to be a useful tool for elucidating the spread dynamics of CRCR-Kp nosocomial strains and could help to limit their diffusion.

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Identification of nsp1 gene as the target of SARS‐CoV‐2 real‐time RT‐PCR using nanopore whole‐genome sequencing

Journal of Medical Virology ◽

10.1002/jmv.26140 ◽

2020 ◽

Vol 92 (11) ◽

pp. 2725-2734 ◽

Cited By ~ 4

Author(s):

Wan‐Mui Chan ◽

Jonathan Daniel Ip ◽

Allen Wing‐Ho Chu ◽

Cyril Chik‐Yan Yip ◽

Lap‐Sum Lo ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Real Time ◽

Genome Sequencing ◽

Whole Genome ◽

Rt Pcr ◽

Nsp1 Gene

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494. Fitness Cost of mcr-1-Mediated Colistin Resistance in Carbapenemase-Producing Klebsiella pneumoniae

Open Forum Infectious Diseases ◽

10.1093/ofid/ofz360.563 ◽

2019 ◽

Vol 6 (Supplement_2) ◽

pp. S241-S241

Author(s):

Yi-Tsung Lin ◽

Yi-Hsiang Cheng ◽

Sheng-Hua Chou ◽

Ying-Chi Huang ◽

Liang Chen

Keyword(s):

Growth Rate ◽

Klebsiella Pneumoniae ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Plasmid Stability ◽

Fatal Case ◽

Fitness Cost ◽

Whole Genome ◽

Colistin Resistance ◽

The Impact

Abstract Background The emergence of mobile colistin resistance gene mcr-1, a plasmid-borne polymyxin resistance mechanism, in carbapenem-resistant Klebsiella pneumoniae is an alarming concern. However, previous studies showed that the acquisition of mcr-1 was associated with a significant biological fitness cost in K. pneumoniae. We aimed to study the impact of mcr-1 on the biological fitness in clinical carbapenemase-producing K. pneumoniae strains. Methods Clinical carbapenemase-producing K. pneumoniae strains were collected consecutively at the Taipei Veterans General Hospital between November 2017 and December 2018. The strain positive for mcr-1 was subjected to whole-genome sequencing to delineate its genomic features. Escherichia coli J53 strain was used as the recipient strain in plasmid conjugation assay and the transconjugants were selected with sodium azide and colistin. Plasmid stability was tested by serial passaging in antibiotic-free LB broth for 28 days. The growth rate was compared between the parental mcr-1-bearing strain and the plasmid-cured strain. Results One ST11 strain isolated from a fatal case with bacteremia (KP2509) was found to harbor blaKPC-2, blaOXA-48, and mcr-1. This strain was resistant to colistin (MIC=8 mg/L) and imipenem (MIC≥16 mg/L). Whole-genome sequencing of KP2509 showed that mcr-1, blaKPC-2 and blaOXA-48 were located on an IncHI-FIB type plasmid of 319 Kb, an IncFII type plasmid of 96 Kb, and an IncL type plasmid of 64 Kb, respectively. Conjugation efficiency of mcr-1-bearing plasmid was 2.24 × 10–4, and the colistin MIC of E. coli J53 transconjugant increased from 0.5 to 8 mg/L. The mcr-1-bearing plasmid in KP2509 showed high plasmid stability, and only ~1% were lost after 27-day passages. The resulting plasmid-cured strain (PC-KP2509) was susceptible to colistin (MIC=0.5 mg/L) and had a similar growth rate to that of parental mcr-1-bearing strain KP2509. Conclusion We identified an ST11 K. pneumoniae strain carrying blaKPC-2, blaOXA-48, and mcr-1 genes causing a fatal bacteremia. The large mcr-1-bearing plasmid confers a moderate level of colistin resistance but without significant biological fitness cost in carbapenemase-producing K. pneumoniae, which could result in a serious threat clinically. Disclosures All authors: No reported disclosures.

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Mobile Colistin Resistance Genetic Determinants of Non-Typhoid Salmonella enterica Isolates from Russia

Microorganisms ◽

10.3390/microorganisms9122515 ◽

2021 ◽

Vol 9 (12) ◽

pp. 2515

Author(s):

Konstantin V. Kuleshov ◽

Anastasia S. Pavlova ◽

Elizaveta D. Shedko ◽

Yulia V. Mikhaylova ◽

Gabriele Margos ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Salmonella Enterica ◽

Diarrheal Disease ◽

Whole Genome ◽

Genetic Determinants ◽

Colistin Resistance ◽

Oxford Nanopore ◽

Susceptible Isolate ◽

Long Read

Polymyxin resistance, determined by mcr genes located on plasmid DNA, currently poses a high epidemiological threat. Non-typhoid Salmonella (NTS) are one of the key pathogens causing diarrheal diseases. Here, we report the isolation and whole genome sequencing of multidrug colistin-resistant/susceptible isolates of non-typhoid Salmonella enterica serovars carrying mcr genes. Non-typhoid strains of Salmonella enterica subsp. enterica were isolated during microbiological monitoring of the environment, food, and diarrheal disease patients between 2018 and 2020 in Russia (n = 586). mcr-1 genes were detected using a previously developed qPCR assay, and whole genome sequencing of mcr positive isolates was performed by both short-read (Illumina) and long-read (Oxford Nanopore) approaches. Three colistin-resistant isolates, including two isolates of S. Enteritidis and one isolate of S. Bovismorbificans, carried the mcr-1.1 gene located on IncX4 and IncI2 conjugative plasmids, respectively. The phenotypically colistin-susceptible isolate of S. Typhimurium carried a mcr-9 gene on plasmid IncHI2. In conclusion, we present the first three cases of mcr gene-carrying NTS isolates detected in Russia with both outbreak and sporadic epidemiological backgrounds.

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The Brazilian Rare Genomes Project: validation of whole genome sequencing for rare diseases diagnosis

10.1101/2021.10.01.21264436 ◽

2021 ◽

Author(s):

Antonio Victor Campos Coelho ◽

Bruna Mascaro Cordeiro de Azevedo ◽

Danielle Ribeiro Lucon ◽

Maria Soares Nobrega ◽

Rodrigo de Souza Reis ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Rare Diseases ◽

Clinical Laboratory ◽

Genomic Medicine ◽

Uniparental Disomy ◽

Public Healthcare ◽

Whole Genome ◽

Single Nucleotide Variants ◽

F Measure

Rare diseases affect 3.2 to 13.2 million individuals in Brazil. The Brazilian Rare Genomes Project is envisioned to further the implementation of genomic medicine into the Brazilian public healthcare system. Here we report the results of the validation of a whole genome sequencing (WGS) procedure for implementation in a clinical laboratory. In addition, we report data quality for the first 1,200 real world patients sequenced. For the validation, we sequenced a well characterized group of 76 samples, including seven gold standard genomes, using a PCR-free WGS protocol on Illumina Novaseq 6000 equipment. We compared the observed variant calls with their expected calls, observing good concordance for single nucleotide variants (SNVs; mean F-measure = 99.82%) and indels (mean F-measure = 99.57%). Copy number variants and structural variants events detection performances were as expected (F-measures 96.6% and 90.3%, respectively). Our protocol presented excellent intra- and inter-assay reproducibility, with coefficients of variation ranging between 0.03% and 0.20% and 0.02% and 0.09%, respectively. Limitations of the procedure include the inability to confidently detect variants such as uniparental disomy, balanced translocations, repeat expansion variants and low-level mosaicism. In summary, the observed performance of the test was in accordance with that seen in the best centers worldwide. The Rare Genomes Project is an important initiative to improve Brazil's general population access to the innovative WGS technology which has the potential to reduce the time until diagnosis of rare diseases, bringing pivotal improvements for the quality of life of the affected individuals.

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