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

2019 ◽  
Vol 57 (6) ◽  
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.

scholarly journals Evolution and Population Dynamics of Clonal Complex 152 Community-Associated Methicillin-Resistant Staphylococcus aureus

mSphere ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Sharmin Baig ◽  
Anders Rhod Larsen ◽  
Patrícia Martins Simões ◽  
Frédéric Laurent ◽  
Thor Bech Johannesen ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Clonal Complex ◽  
Whole Genome ◽  
Methicillin Resistant ◽  
Content Type ◽  
Type V ◽  
Panton Valentine Leukocidin ◽  
Valentine Leukocidin

ABSTRACT Since the late 1990s, changes in the epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) were recognized with the emergence of community-associated MRSA (CA-MRSA). CA-MRSA belonging to clonal complex 152 (CC152), carrying the small staphylococcal cassette chromosome mec (SCCmec) type V and encoding the Panton-Valentine leukocidin (PVL), has been observed in Europe. The aim of this study was to investigate its origin, evolution, and dissemination. Whole-genome sequencing was performed on a global collection of 149 CC152 isolates spanning 20 years (93 methicillin-susceptible S. aureus [MSSA] and 56 MRSA isolates). Core genome phylogeny, Bayesian inference, in silico resistance analyses, and genomic characterization were applied. Phylogenetic analysis revealed two major distinct clades, one dominated by MSSA and the other populated only by MRSA. The MSSA isolates were predominately from sub-Saharan Africa, whereas MRSA was almost exclusively from Europe. The European MRSA isolates all harbored an SCCmec type V (5C2&5) element, whereas other SCCmec elements were sporadically detected in MRSA from the otherwise MSSA-dominated clade, including SCCmec types IV (2B), V (5C2), and XIII (9A). In total, 93% of the studied CC152 isolates were PVL positive. Bayesian coalescent inference suggests an emergence of the European CC152-MRSA in the 1990s, while the CC152 lineage dates back to the 1970s. The CA-MRSA CC152 clone mimics the European CC80 CA-MRSA lineage by its emergence from a PVL-positive MSSA ancestor from North Africa or Europe. The CC152 lineage has acquired SCCmec several times, but acquisition of SCCmec type V (5C2&5) seems associated with expansion of MRSA CC152 in Europe. IMPORTANCE Understanding the evolution of CA-MRSA is important in light of the increasing importance of this reservoir in the dissemination of MRSA. Here, we highlight the story of the CA-MRSA CC152 lineage using whole-genome sequencing on an international collection of CC152. We show that the evolution of this lineage is novel and that antibiotic usage may have the potential to select for the phage-encoded Panton-Valentine leukocidin. The diversity of the strains correlated highly to geography, with higher level of resistance observed among the European MRSA isolates. The mobility of the SCCmec element is mandatory for the emergence of novel MRSA lineages, and we show here distinct acquisitions, one of which is linked to the successful clone found throughout Europe today.

scholarly journals Mutation of RNA Polymerase β-Subunit Gene Promotes Heterogeneous-to-Homogeneous Conversion of β-Lactam Resistance in Methicillin-Resistant Staphylococcus aureus

2013 ◽  
Vol 57 (10) ◽  
pp. 4861-4871 ◽  
Author(s):  
Yoshifumi Aiba ◽  
Yuki Katayama ◽  
Tomomi Hishinuma ◽  
Hiroko Murakami-Kuroda ◽  
Longzhu Cui ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Rna Polymerase ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Phenotypic Expression ◽  
Amino Acid Replacement ◽  
Whole Genome ◽  
Β Subunit ◽  
Methicillin Resistant ◽  
Content Type

ABSTRACTThree types of phenotypic expression of β-lactam resistance have been reported in methicillin-resistantStaphylococcus aureus(MRSA): heterogeneous, homogeneous, and Eagle-type resistance. Heterogeneous-to-homogeneous conversion of β-lactam resistance is postulated to be caused by a chromosomal mutation (chr*) in addition to the expression of themecAgene. Eagle-type resistance is a unique phenotype ofchr*occurring in pre-MRSA strain N315 whosemecAgene expression is strongly repressed by an intactmecIgene. We here report that certain mutations of therpoBgene, encoding the RNA polymerase β subunit, belong tochr*. We studied homogeneous MRSA (homo-MRSA) strain N315ΔIP-H5 (abbreviated as ΔIP-H5), which was obtained from hetero-MRSA strain N315ΔIP by selection with 8 mg/liter imipenem. Whole-genome sequencing of ΔIP-H5 revealed the presence of a unique mutation in therpoBgene,rpoB(N967I), causing the amino acid replacement of Asn by Ile at position 967 of RpoB. The effect of therpoB(N967I) mutation was confirmed by constructing a revertant H5rpoB(I967N) strain as well as an N315-derived mutant, N315rpoB(N967I). H5rpoB(I967N) regained the hetero-resistance phenotype, and the N315rpoB(N967I) strain showed an Eagle-type phenotype similar to that of the typical Eagle-type MRSA strain N315h4. Furthermore, subsequent whole-genome sequencing revealed that N315h4 also had a missense mutation ofrpoB(R644H). Introduction of therpoB(N967I) mutation was accompanied by decreased autolysis, prolonged doubling time, and tolerance to bactericidal concentrations of methicillin. We consider thatrpoBmutations are the major cause for heterogeneous-to-homogeneous phenotypic conversion of β-lactam resistance in MRSA strain N315 and its derived strains.

scholarly journals Enhanced Tracking of Nosocomial Transmission of Endemic Sequence Type 22 Methicillin-Resistant Staphylococcus aureus Type IV Isolates among Patients and Environmental Sites by Use of Whole-Genome Sequencing

2015 ◽  
Vol 54 (2) ◽  
pp. 445-448 ◽  
Author(s):  
Peter M. Kinnevey ◽  
Anna C. Shore ◽  
Micheál Mac Aogáin ◽  
Eilish Creamer ◽  
Gráinne I. Brennan ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Sequence Type ◽  
Hospital Environment ◽  
Whole Genome ◽  
Single Nucleotide Variants ◽  
Methicillin Resistant ◽  
Content Type ◽  
Type Iv

Whole-genome sequencing (WGS) of 41 patient and environmental sequence type 22 methicillin-resistantStaphylococcus aureusstaphylococcal cassette chromosomemectype IV (ST22-MRSA-IV) isolates recovered over 6 weeks in one acute hospital ward in Dublin, Ireland, where ST22-MRSA IV is endemic, revealed 228 pairwise combinations differing by <40 single nucleotide variants corresponding to potential cross-transmission events (CTEs). In contrast, 15 pairwise combinations of isolates representing five CTEs were previously identified by conventional molecular epidemiological typing. WGS enhanced ST22-MRSA-IV tracking and highlighted potential transmission of MRSA via the hospital environment.

scholarly journals A Staphylococcus xylosus Isolate with a NewmecCAllotype

2012 ◽  
Vol 57 (3) ◽  
pp. 1524-1528 ◽  
Author(s):  
Ewan M. Harrison ◽  
Gavin K. Paterson ◽  
Matthew T. G. Holden ◽  
Fiona J. E. Morgan ◽  
Anders Rhod Larsen ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Whole Genome ◽  
Content Type ◽  
Staphylococcus Xylosus ◽  
Novel Variant ◽  
Staphylococcal Cassette Chromosome ◽  
Class E

ABSTRACTRecently, a novel variant ofmecAknown asmecC(mecALGA251) was identified inStaphylococcus aureusisolates from both humans and animals. In this study, we identified aStaphylococcus xylosusisolate that harbors a new allotype of themecCgene,mecC1. Whole-genome sequencing revealed thatmecC1forms part of a class Emeccomplex (mecI-mecR1-mecC1-blaZ) located at theorfXlocus as part of a likely staphylococcal cassette chromosomemecelement (SCCmec) remnant, which also contains a number of other genes present on the type XI SCCmec.

scholarly journals Detecting Staphylococcus aureus Virulence and Resistance Genes: a Comparison of Whole-Genome Sequencing and DNA Microarray Technology

2016 ◽  
Vol 54 (4) ◽  
pp. 1008-1016 ◽  
Author(s):  
Lena Strauß ◽  
Ulla Ruffing ◽  
Salim Abdulla ◽  
Abraham Alabi ◽  
Ruslan Akulenko ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Dna Microarray ◽  
Resistance Genes ◽  
In Silico ◽  
Target Genes ◽  
Whole Genome ◽  
Microarray Hybridization ◽  
Content Type

Staphylococcus aureusis a major bacterial pathogen causing a variety of diseases ranging from wound infections to severe bacteremia or intoxications. Besides host factors, the course and severity of disease is also widely dependent on the genotype of the bacterium. Whole-genome sequencing (WGS), followed by bioinformatic sequence analysis, is currently the most extensive genotyping method available. To identify clinically relevant staphylococcal virulence and resistance genes in WGS data, we developed anin silicotyping scheme for the software SeqSphere+(Ridom GmbH, Münster, Germany). The implemented target genes (n= 182) correspond to those queried by the IdentibacS. aureusGenotyping DNA microarray (Alere Technologies, Jena, Germany). Thein silicoscheme was evaluated by comparing the typing results of microarray and of WGS for 154 humanS. aureusisolates. A total of 96.8% (n= 27,119) of all typing results were equally identified with microarray and WGS (40.6% present and 56.2% absent). Discrepancies (3.2% in total) were caused by WGS errors (1.7%), microarray hybridization failures (1.3%), wrong prediction of ambiguous microarray results (0.1%), or unknown causes (0.1%). Superior to the microarray, WGS enabled the distinction of allelic variants, which may be essential for the prediction of bacterial virulence and resistance phenotypes. Multilocus sequence typing clonal complexes and staphylococcal cassette chromosomemecelement types inferred from microarray hybridization patterns were equally determined by WGS. In conclusion, WGS may substitute array-based methods due to its universal methodology, open and expandable nature, and rapid parallel analysis capacity for different characteristics in once-generated sequences.

scholarly journals Unsuspected clonal spread of Methicillin-resistant Staphylococcus aureus causing bloodstream infections in hospitalized adults detected using whole genome sequencing

2021 ◽  
Author(s):  
Brooke Morgan Talbot ◽  
Natasia F Jacko ◽  
Robert A Petit ◽  
David A Pegues ◽  
Margot J Shumaker ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
Bloodstream Infections ◽  
Whole Genome ◽  
Nucleotide Polymorphisms ◽  
Methicillin Resistant ◽  
Clonal Spread ◽  
Epidemiological Characteristics

Background: Though detection of transmission clusters of methicillin-resistant Staphylococcus aureus (MRSA) infections is a priority for infection control personnel in hospitals, the transmission dynamics of MRSA among hospitalized patients with bloodstream infections (BSIs) has not been thoroughly studied. Whole genome sequencing (WGS) of MRSA isolates for surveillance is valuable for detecting outbreaks in hospitals, but the bioinformatic approaches used are diverse and difficult to compare. Methods: We combined short-read WGS with genotypic, phenotypic, and epidemiological characteristics of 106 MRSA BSI isolates collected for routine microbiological diagnosis from inpatients in two hospitals over 12 months. Clinical data and hospitalization history were abstracted from electronic medical records. We compared three genome sequence alignment strategies to assess similarity in cluster ascertainment. We conducted logistic regression to measure the probability of predicting prior hospital overlap between clustered patient isolates by the genetic distance of their isolates. Results: While the three alignment approaches detected similar results, they showed some variation. A pangenome-based alignment method was most consistent across MRSA clonal complexes. We identified nine unique clusters of closely-related BSI isolates. Most BSI were healthcare-associated and community-onset. Our logistic model showed that with 13 single nucleotide polymorphisms the likelihood that any two patients in a cluster overlapped in a hospital was 50 percent. Conclusions: Multiple clusters of closely related MRSA isolates can be identified using WGS among strains cultured from BSI in two hospitals. Genomic clustering of these infections suggest that transmission resulted from a mix of community spread and healthcare exposures long before BSI diagnosis.

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