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

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.

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Comparative Whole-Genome Phylogeny of Animal, Environmental, and Human Strains Confirms the Genogroup Organization and Diversity of the Stenotrophomonas maltophilia Complex

Applied and Environmental Microbiology ◽

10.1128/aem.02919-19 ◽

2020 ◽

Vol 86 (10) ◽

Author(s):

Mélanie Mercier-Darty ◽

Guilhem Royer ◽

Brigitte Lamy ◽

Chadly Charron ◽

Olivier Lemenand ◽

...

Keyword(s):

Genetic Diversity ◽

Antimicrobial Resistance ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Resistance Genes ◽

Stenotrophomonas Maltophilia ◽

Whole Genome ◽

Content Type ◽

Genetic Organization ◽

Animal Strains

ABSTRACT The Stenotrophomonas maltophilia complex (Smc) comprises opportunistic environmental Gram-negative bacilli responsible for a variety of infections in both humans and animals. Beyond its large genetic diversity, its genetic organization in genogroups was recently confirmed through the whole-genome sequencing of human and environmental strains. As they are poorly represented in these analyses, we sequenced the whole genomes of 93 animal strains to determine their genetic background and characteristics. Combining these data with 81 newly sequenced human strains and the genomes available from RefSeq, we performed a genomic analysis that included 375 nonduplicated genomes with various origins (animal, 104; human, 226; environment, 30; unknown, 15). Phylogenetic analysis and clustering based on genome-wide average nucleotide identity confirmed and specified the genetic organization of Smc in at least 20 genogroups. Two new genogroups were identified, and two previously described groups were further divided into two subgroups each. Comparing the strains isolated from different host types and their genogroup affiliation, we observed a clear disequilibrium in certain groups. Surprisingly, some antimicrobial resistance genes, integrons, and/or clusters of attC sites lacking integron-integrase (CALIN) sequences targeting antimicrobial compounds extensively used in animals were mainly identified in animal strains. We also identified genes commonly found in animal strains coding for efflux systems. The result of a large whole-genome analysis performed by us supports the hypothesis of the putative contribution of animals as a reservoir of Stenotrophomonas maltophilia complex strains and/or resistance genes for strains in humans. IMPORTANCE Given its naturally large antimicrobial resistance profile, the Stenotrophomonas maltophilia complex (Smc) is a set of emerging pathogens of immunosuppressed and cystic fibrosis patients. As it is group of environmental microorganisms, this adaptation to humans is an opportunity to understand the genetic and metabolic selective mechanisms involved in this process. The previously reported genomic organization was incomplete, as data from animal strains were underrepresented. We added the missing piece of the puzzle with whole-genome sequencing of 93 strains of animal origin. Beyond describing the phylogenetic organization, we confirmed the genetic diversity of the Smc, which could not be estimated through routine phenotype- or matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF)-based laboratory tests. Animals strains seem to play a key role in the diversity of Smc and could act as a reservoir for mobile resistance genes. Some genogroups seem to be associated with particular hosts; the genetic support of this association and the role of the determinants/corresponding genes need to be explored.

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Frequency of Antimicrobial Resistance Genes in Salmonella From Brazil by in silico Whole-Genome Sequencing Analysis: An Overview of the Last Four Decades

Frontiers in Microbiology ◽

10.3389/fmicb.2020.01864 ◽

2020 ◽

Vol 11 ◽

Author(s):

Grazielle Lima Rodrigues ◽

Pedro Panzenhagen ◽

Rafaela Gomes Ferrari ◽

Anamaria dos Santos ◽

Vania Margaret Flosi Paschoalin ◽

...

Keyword(s):

Antimicrobial Resistance ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Resistance Genes ◽

In Silico ◽

Whole Genome ◽

Sequencing Analysis ◽

Antimicrobial Resistance Genes

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Characterization of SXT/R391 Integrative and Conjugative Elements in Proteus mirabilis Isolates from Food-Producing Animals in China

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02852-15 ◽

2016 ◽

Vol 60 (3) ◽

pp. 1935-1938 ◽

Cited By ~ 25

Author(s):

Chang-Wei Lei ◽

An-Yun Zhang ◽

Hong-Ning Wang ◽

Bi-Hui Liu ◽

Li-Qin Yang ◽

...

Keyword(s):

Escherichia Coli ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Resistance Genes ◽

Proteus Mirabilis ◽

Whole Genome ◽

Content Type ◽

Integrative And Conjugative Elements ◽

Animal Farms

SXT/R391 integrative and conjugative elements (ICEs) were detected in 8 out of 125Proteus mirabilisisolates from food-producing animals in China. Whole-genome sequencing revealed that seven ICEs were identical to ICEPmiJpn1, carrying the cephalosporinase geneblaCMY-2. Another one, designated ICEPmiChn1, carried five resistance genes. All eight ICEs could be transferred toEscherichia colivia conjugation. The results highlight the idea that animal farms are important reservoir of the SXT/R391 ICE-containingP. mirabilis.

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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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A Staphylococcus xylosus Isolate with a NewmecCAllotype

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01882-12 ◽

2012 ◽

Vol 57 (3) ◽

pp. 1524-1528 ◽

Cited By ~ 49

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.

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Whole-Genome Sequencing Analysis Accurately Predicts Antimicrobial Resistance Phenotypes in Campylobacter spp.

Applied and Environmental Microbiology ◽

10.1128/aem.02873-15 ◽

2015 ◽

Vol 82 (2) ◽

pp. 459-466 ◽

Cited By ~ 132

Author(s):

S. Zhao ◽

G. H. Tyson ◽

Y. Chen ◽

C. Li ◽

S. Mukherjee ◽

...

Keyword(s):

Antimicrobial Resistance ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Resistance Genes ◽

Antimicrobial Agents ◽

The United States ◽

23S Rrna ◽

Whole Genome ◽

Sequencing Analysis ◽

Content Type

ABSTRACTThe objectives of this study were to identify antimicrobial resistance genotypes forCampylobacterand to evaluate the correlation between resistance phenotypes and genotypes usingin vitroantimicrobial susceptibility testing and whole-genome sequencing (WGS). A total of 114Campylobacterspecies isolates (82C. coliand 32C. jejuni) obtained from 2000 to 2013 from humans, retail meats, and cecal samples from food production animals in the United States as part of the National Antimicrobial Resistance Monitoring System were selected for study. Resistance phenotypes were determined using broth microdilution of nine antimicrobials. Genomic DNA was sequenced using the Illumina MiSeq platform, and resistance genotypes were identified using assembled WGS sequences through blastx analysis. Eighteen resistance genes, includingtet(O),blaOXA-61,catA,lnu(C),aph(2″)-Ib,aph(2″)-Ic,aph(2′)-If,aph(2″)-Ig,aph(2″)-Ih,aac(6′)-Ie-aph(2″)-Ia,aac(6′)-Ie-aph(2″)-If,aac(6′)-Im,aadE,sat4,ant(6′),aad9,aph(3′)-Ic, andaph(3′)-IIIa, and mutations in two housekeeping genes (gyrAand 23S rRNA) were identified. There was a high degree of correlation between phenotypic resistance to a given drug and the presence of one or more corresponding resistance genes. Phenotypic and genotypic correlation was 100% for tetracycline, ciprofloxacin/nalidixic acid, and erythromycin, and correlations ranged from 95.4% to 98.7% for gentamicin, azithromycin, clindamycin, and telithromycin. All isolates were susceptible to florfenicol, and no genes associated with florfenicol resistance were detected. There was a strong correlation (99.2%) between resistance genotypes and phenotypes, suggesting that WGS is a reliable indicator of resistance to the nine antimicrobial agents assayed in this study. WGS has the potential to be a powerful tool for antimicrobial resistance surveillance programs.

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Genomically Informed Surveillance for Carbapenem-Resistant Enterobacteriaceae in a Health Care System

mBio ◽

10.1128/mbio.01030-15 ◽

2015 ◽

Vol 6 (4) ◽

Cited By ~ 60

Author(s):

Nicole D. Pecora ◽

Ning Li ◽

Marc Allard ◽

Cong Li ◽

Esperanza Albano ◽

...

Keyword(s):

Health Care ◽

Drug Resistance ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Resistance Genes ◽

Enterobacter Cloacae ◽

Mobile Elements ◽

Whole Genome ◽

Content Type ◽

Carbapenem Resistant

ABSTRACT Carbapenem-resistant Enterobacteriaceae (CRE) are an urgent public health concern. Rapid identification of the resistance genes, their mobilization capacity, and strains carrying them is essential to direct hospital resources to prevent spread and improve patient outcomes. Whole-genome sequencing allows refined tracking of both chromosomal traits and associated mobile genetic elements that harbor resistance genes. To enhance surveillance of CREs, clinical isolates with phenotypic resistance to carbapenem antibiotics underwent whole-genome sequencing. Analysis of 41 isolates of Klebsiella pneumoniae and Enterobacter cloacae, collected over a 3-year period, identified K. pneumoniae carbapenemase (KPC) genes encoding KPC-2, −3, and −4 and OXA-48 carbapenemases. All occurred within transposons, including multiple Tn4401 transposon isoforms, embedded within more than 10 distinct plasmids representing incompatibility (Inc) groups IncR, -N, -A/C, -H, and -X. Using short-read sequencing, draft maps were generated of new KPC-carrying vectors, several of which were derivatives of the IncN plasmid pBK31551. Two strains also had Tn4401 chromosomal insertions. Integrated analyses of plasmid profiles and chromosomal single-nucleotide polymorphism (SNP) profiles refined the strain patterns and provided a baseline hospital mobilome to facilitate analysis of new isolates. When incorporated with patient epidemiological data, the findings identified limited outbreaks against a broader 3-year period of sporadic external entry of many different strains and resistance vectors into the hospital. These findings highlight the utility of genomic analyses in internal and external surveillance efforts to stem the transmission of drug-resistant strains within and across health care institutions. IMPORTANCE We demonstrate how detection of resistance genes within mobile elements and resistance-carrying strains furthers active surveillance efforts for drug resistance. Whole-genome sequencing is increasingly available in hospital laboratories and provides a powerful and nuanced means to define the local landscape of drug resistance. In this study, isolates of Klebsiella pneumoniae and Enterobacter cloacae with resistance to carbapenem antibiotics were sequenced. Multiple carbapenemase genes were identified that resided in distinct transposons and plasmids. This mobilome, or population of mobile elements capable of mobilizing drug resistance, further highlighted the degree of strain heterogeneity while providing a detailed timeline of carbapenemase entry into the hospital over a 3-year period. These surveillance efforts support effective targeting of infection control resources and the development of institution-specific repositories of resistance genes and the mobile elements that carry them.

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Isolation, Whole-Genome Sequencing, and Annotation of Three Unclassified Antibiotic-Producing Bacteria, Enterobacter sp. Strain RIT 637, Pseudomonas sp. Strain RIT 778, and Deinococcus sp. Strain RIT 780

Microbiology Resource Announcements ◽

10.1128/mra.00863-21 ◽

2021 ◽

Vol 10 (48) ◽

Author(s):

Marissa N. Schroeter ◽

Safiya J. Gazali ◽

Anutthaman Parthasarathy ◽

Crista B. Wadsworth ◽

Renata Rezende Miranda ◽

...

Keyword(s):

Escherichia Coli ◽

Staphylococcus Aureus ◽

Pseudomonas Aeruginosa ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Disk Diffusion ◽

Whole Genome ◽

Pseudomonas Sp ◽

Content Type

We report the isolation, whole-genome sequencing, and annotation of Enterobacter sp. strain RIT 637, Pseudomonas sp. strain RIT 778, and Deinococcus sp. strain RIT 780. Disk diffusion assays using spent medium demonstrated that all bacteria produced bactericidal compounds against Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Staphylococcus aureus ATCC 25923.

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

mSphere ◽

10.1128/msphere.00226-20 ◽

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.

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Exploiting Bacterial Whole-Genome Sequencing Data for Evaluation of Diagnostic Assays: Campylobacter Species Identification as a Case Study

Journal of Clinical Microbiology ◽

10.1128/jcm.01522-16 ◽

2016 ◽

Vol 54 (12) ◽

pp. 2882-2890 ◽

Cited By ~ 9

Author(s):

Melissa J. Jansen van Rensburg ◽

Craig Swift ◽

Alison J. Cody ◽

Claire Jenkins ◽

Martin C. J. Maiden

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Clinical Microbiology ◽

Target Genes ◽

Molecular Diagnostic ◽

Whole Genome Sequencing Data ◽

Whole Genome ◽

Rt Pcr ◽

Content Type ◽

Diagnostic Assays

The application of whole-genome sequencing (WGS) to problems in clinical microbiology has had a major impact on the field. Clinical laboratories are now using WGS for pathogen identification, antimicrobial susceptibility testing, and epidemiological typing. WGS data also represent a valuable resource for the development and evaluation of molecular diagnostic assays, which continue to play an important role in clinical microbiology. To demonstrate this application of WGS, this study used publicly available genomic data to evaluate a duplex real-time PCR (RT-PCR) assay that targetsmapAandceuEfor the detection ofCampylobacter jejuniandCampylobacter coli, leading global causes of bacterial gastroenteritis.In silicoanalyses ofmapAandceuEprimer and probe sequences from 1,713 genetically diverseC. jejuniandC. coligenomes, supported by RT-PCR testing, indicated that the assay was robust, with 1,707 (99.7%) isolates correctly identified. The high specificity of themapA-ceuEassay was the result of interspecies diversity and intraspecies conservation of the target genes inC. jejuniandC. coli. Rare instances of a lack of specificity amongC. coliisolates were due to introgression inmapAor sequence diversity inceuE. The results of this study illustrate how WGS can be exploited to evaluate molecular diagnostic assays by using publicly available data, online databases, and open-source software.

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