Genetic Platforms Research Articles

Occurrence of carbapenemase-producing Enterobacteriaceae in a Portuguese river: blaNDM, blaKPC and blaGES among the detected genes

Carbapenems are used as last-resort drugs to treat infections caused by multidrug-resistant bacteria. Despite the increasing number of reports of carbapenem-resistant Enterobacteriaceae (CRE), there is still limited information on their distribution or prevalence in the environment. Our aim was to assess the occurrence of CRE in the Lis river (Portugal) and to characterize the genetic platforms linked to carbapenemase genes. We collected six water samples from sites near a wastewater treatment plant (n = 4 samples) and livestock farms (n = 2). Twenty-four CRE were characterized by BOX element-polymerase chain reaction (BOX-PCR), and thirteen representative isolates were analysed by Pulsed-Field Gel Electrophoresis (PFGE) and by sequencing the 16S rRNA gene. Antimicrobial susceptibility testing, PCR screening for carbapenemase-encoding genes, conjugation experiments and plasmid analysis were performed. Four isolates were chosen for whole-genome sequencing. All water samples contained CRE (4.0 CFU/mL on average). Representative isolates were multidrug-resistant (resistant to ciprofloxacin, trimethoprim-sulfamethoxazole and to all β-lactams tested) and were identified as K. pneumoniae, Enterobacter and Citrobacter. Isolates carried plasmids and harboured carbapenemase-encoding genes: blaKPC-3 in K. pneumoniae (n = 9), blaNDM-1 in Enterobacter (n = 3) and blaGES-5 in Citrobacter (n = 1). Conjugation experiments were successful in two Klebsiella isolates. Enterobacter PFGE profiles grouped in one cluster while Klebsiella were divided in three clusters and a singleton. Whole-genome sequencing analysis revealed blaGES-5 within a novel class 3 integron (In3-16) located on an IncQ/pQ7-like plasmid in Citrobacter freundii CR16. blaKPC-3 was present on IncFIA-FII pBK30683-like plasmids, which were subsequently confirmed in all K. pneumoniae (n = 9). Furthermore, blaKPC-3 was part of a genomic island in K. pneumoniae CR12. In E. roggenkampii CR11, blaNDM-1 was on an IncA/C2 plasmid. The carbapenemase-encoding plasmids harboured other resistance determinants and mobile genetic elements. Our results demonstrate that Lis river is contaminated with CRE, highlighting the need for monitoring antibiotic resistance in aquatic environments, especially to last-resort drugs.

Conjugation Protocol Optimised for Roseburia inulinivorans and Eubacterium rectale.

Roseburia and Eubacterium species of the human gut microbiota play an important role in the maintaince of human health, partly by producing butyrate, the main energy source of our colonic epithelial cells. However, our knowledge of the biochemistry and physiology of these bacteria has been limited by a lack of genetic manipulation techniques. Conjugative transposons previously introduced into Roseburia species could not be easily modified, greatly limiting their applicability as genetic modification platforms. Modular plasmid shuttle vectors have previously been developed for Clostridium species, which share a taxonomic order with Roseburia and Eubacterium, raising the possibility that these vectors could be used in these organisms. Here, we describe an optimized conjugation protocol enabling the transfer of autonomously replicating plasmids from an E. coli donor strain into Roseburia inulinivorans and Eubacterium rectale. The modular nature of the plasmids and their ability to be maintained in the recipient bacterium by autonomous replication makes them ideal for investigating heterologous gene expression, and as a platform for other genetic tools including antisense RNA silencing or mobile group II interon gene disruption strategies.

Molecular characteristics of oxazolidinone resistance in enterococci from a multicenter study in China

BackgroundLinezolid-resistant enterococci pose great challenges in clinical practice. The aim of this study is to study the mechanisms underlying the resistance and genetic environment of antimicrobial resistance gene of linezolid-resistant enterococci.ResultsThe linezolid MICs of 16 enterococci were 4 mg/L to 16 mg/L. Four strains belonged to multi-drug resistant (MDR) bacteria. The sequence types (STs) of 13 enterococci strains performed WGS were diverse: 3 ST476, 1 ST86, ST116, ST480, ST59, ST416, ST21, ST67, ST16, ST585 and ST18. None of them carried multi-drug resistance gene cfr. Only one strain had the G2658 T mutation of target 23S rRNA gene. Thirteen (13/16, 81.3%) strains harbored the novel oxazolidinone resistance gene optrA. WGS analysis showed that the optrA gene was flanked by sequence IS1216E insertion in 13 strains, and optrA was adjacent to transposons Tn558 in two strains and Tn554 in one strain. The optrA gene was identified to be co-localized with fexA, the resistance genes mediated florfenicol resistance in 13 strains, and ermA1, the resistance genes mediated erythromycin resistance in 9 strains, indicating that linezolid-resistant strains may be selected due to non-oxazolidinone antibiotics (i.e. macrolides and florfenicol) usage.ConclusionOur findings demonstrate the high diversity of optrA-carrying genetic platforms. The mobile genetic elements (MGEs) may play an important role in the dissemination of optrA into the enterococci isolates of human origin. The genetic evidence of transferable feature and co-selection of optrA should be gave more attention in clinical practice.

Genome and plasmid context of two rmtG-carrying Enterobacter hormaechei isolated from urinary tract infections in Brazil.

Enterobacter hormaechei is an important causative agent of severe infections in critically ill patients. Aminoglycosides are among the main antibiotics for the treatment of E. hormaechei infections, however the development of antimicrobial resistance is an increasing problem. RmtG is a 16S rRNA methyltransferase, a class of enzymes conferring high-level resistance to clinically relevant aminoglycosides. The aim of this study was to characterise the full genetic context of plasmids harbouring the rmtG gene in two aminoglycoside-resistant E. hormaechei isolated in Brazil. ThermtG-harbouring plasmids were transferred to an Escherichia coli J53 recipient strain and were fully sequenced using a MiSeq sequencing system. Complete genome assemblies were accomplished using a combination of Newbler v.3.0, SPAdes 3.10.0 and phrap/cross_match programs. Plasmid sequences were annotated using RAST server and were then manually curated using BLAST databases and ISfinder. Easyfig 2.0 was used to map and compare regions of interest containing rmtG in both plasmids. Both isolates carried thermtG gene on an IncA/C plasmid of ˜152kb and ˜235kb, respectively, associated with a Tn3 transposon. The plasmids contain a transfer region as well as genes involved in plasmid stability and resistance to β-lactams, sulfonamides and quaternary ammonium compounds. One of the plasmids also carried the mrk operon encoding type 3 fimbriae. This first detection ofrmtG in E. hormaechei supports the ability for horizontal transfer. The location in complex genetic platforms carried by Tn3 transposons in IncA/C plasmids may facilitate dissemination to other Gram-negative pathogens, further limiting treatment options.

Using genomic data for selecting the treatment of lymphoma patients.

Genomic profiling platforms provide unprecedented genetic information of lymphoma biology, yet information has yet to be readily integrated into clinical medicine. This review summarizes the important concepts of utilizing genomics to aide disease management. A wide range of clinical grade genetic sequencing platforms are available, therefore the selection of sequencing platform should ideally be based on biological and clinical questions, as well as the strength and weaknesses of individual platform. Different evidence-based guidelines exist to aide clinical judgment; however, few have well curated, easy to search platforms. Using one guideline proposed by several regulatory groups, our review summarizes genetic alterations with diagnostic, prognostic and therapeutic potential in the major subtypes of lymphoma. A comprehensive database of genetic alterations that contribute to clinical care in lymphoma is needed. Ideally, a database which accounts for single and pathway-based genetic alterations may be developed to guide development and interventions for management of lymphoma.

Changing epidemiology of KPC-producing Klebsiella pneumoniae in Argentina: Emergence of hypermucoviscous ST25 and high-risk clone ST307.

To assess the epidemiological features of 76 Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae (KPC-Kp) isolates recovered from three hospitals in Buenos Aires, Argentina, during 2015-2017. Antimicrobial susceptibilities were determined according to CLSI Clinical and Laboratoy Standards guidelines. Molecular typing of KPC-Kp was performed by pulsed-field gel electrophoresis (PFGE)-Xbal and multilocus sequence typing. Plasmid encoded genes involved in carbapenem, fosfomycin and colistin resistance were detected by polymerase chain reaction (PCR) and sequencing. Also, mgrB inactivation was investigated in those colistin-resistant isolates. Genetic platforms involved in horizontal spread of blaKPC were investigated by PCR mapping. Besides β-lactams, high resistance rates were observed for gentamycin, quinolones and trimethoprim-sulfamethoxazole. KPC-Kp sequence type (ST)258 corresponded to 26% of the isolates, while 42% corresponded to ST25. The other isolates were distributed in a diversity of lineages such as ST11 (10.5%), ST392 (10.5%), ST307, ST13, ST101, ST15 and ST551. blaKPC-2 was detected in 75 of 76 isolates, and one ST307 isolate harboured blaKPC-3. Tn4401 was identified as the genetic platform for blaKPC in epidemic lineages such as ST258 and ST307. However, in ST25 and ST392, which are usually not related to blaKPC, a blaKPC-bearing non-Tn4401 element was identified. Alterations in mgrB were detected in seven of 11 colistin-resistant isolates. Despite previous reports in Argentina, ST258 is no longer the absolute clone among KPC-Kp isolates. In the present study, dissemination of more virulent lineages such as the hypermucoviscous ST25 was detected. The emergence of the high-risk clone ST307 and occurrence of blaKPC-3 was noticed for the first time in this region.

191-OR: New Regulators of Insulin-Stimulated GLUT4 Exocytosis

Insulin promotes glucose uptake by triggering the translocation of the glucose transporter GLUT4 from intracellular storage vesicles to the cell surface through exocytosis. Defects in GLUT4 exocytosis are a hallmark of insulin resistance (IR) and type 2 diabetes (T2D). To develop effective and safe treatments for IR and T2D, it is crucial to gain a comprehensive understanding of insulin-stimulated GLUT4 exocytosis at the molecular level. Recently, our group developed new genetic screen platforms to systematically dissect insulin-stimulated GLUT4 exocytosis, taking advantage of the revolutionary CRISPR-Cas9 genome editing system. Our genome-wide CRISPR screens recovered known GLUT4 exocytic regulators but most of the hits were not previously linked to the GLUT4 exocytic pathway. Here, I will focus on AAGAB, a negative regulator of GLUT4 translocation identified in the screens. We discovered that AAGAB plays a dual role in GLUT4 trafficking: 1) it promotes the endocytosis of GLUT4, and 2) it negatively regulates the SNARE-Munc18-mediated GLUT4 vesicle fusion. In addition, I will discuss OSBPL8 and OSBPL10, another two negative regulators identified in our screens. OSBPL8 and OSBPL10 are putative lipid transfer proteins operating between intracellular organelles. We propose that OSBPL8 and OSBPL10 regulate lipid dynamics required for insulin signaling, thereby influencing the activities of GLUT4 exocytic regulators. These genetic studies will facilitate the identification of new therapeutic targets for IR and T2D. Disclosure J. Shen: None. Funding National Institutes of Health

Systematic Review of Hearing Loss Genes in the African American Population.

Literature review of the genetic etiology of hearing loss (HL) in the African American (AA) population. PubMed, EBSCO, and CINAHL were accessed from 1966 to 2018. PRISMA guidelines were followed. Search terms included permutations of "hearing loss," "African American," "black," and "genetic"; "African American" was then cross-referenced against documented HL genes. AA subjects included in multiethnic cohorts of genetic HL testing were identified by searching the key terms "hearing loss" and "ethnic cohort" and "genetic." The Q-Genie tool was used in the quality assessment of included studies. An allele frequency meta-analysis of pathogenic GJB2 variants in the AA population was performed and stratified by hearing status. Four hundred seventeen articles were reviewed, and 26 met our inclusion criteria. Ten studies were included in the GJB2 meta-analysis. In the general AA population, pathogenic GJB2 variants are rare, including the 35delG allele, which displayed a carrier frequency of 0.05%. Pathogenic variants were discovered in seven nonsyndromic HL genes (GJB2, MYO3A, TECTA, STRC, OTOF, MYH14, TMC1), eight syndromic HL genes, and one mitochondrial HL gene. Recent comprehensive genetic testing using custom genetic HL testing platforms has yielded only a 26% molecular diagnosis rate for HL etiologies in the AA population. Investigators should be encouraged to provide an ethnic breakdown of results. Sparse literature and poor diagnosis rates indicate that genes involved in HL in the AA population have yet to be identified. Future explorative investigations using next-generation sequencing technologies, such as whole-exome sequencing, into the AA population are warranted.

Novel patterns in the molecular epidemiology of KPC-producing Klebsiella pneumoniae in Tucumán, Argentina.

In Argentina, there has been an abrupt increase in KPC-2-producing Klebsiella pneumoniae (K. pneumoniae). Tucumán is a multi-border area, so the rapid dissemination of carbapenem-resistant K. pneumoniae is a clinically relevant problem for the region. This study aimed to investigate the epidemiological and molecular patterns of KPC-producing K. pneumoniae clinical isolates collected from different hospitals in Tucumán. Carbapenem-resistant K. pneumoniae strains were sequentially and uniquely collected during two time periods. Antibiotic susceptibility was determined by the automated Vitex 2® system and using the standard agar dilution test. Multilocus sequence typing and pulsed-field electrophoresis were used for epidemiological analysis. The genetic structures around blaKPC and the encoding genes of extended-spectrum β-lactamases were detected by polymerase chain reaction and sequencing. Plasmids were analysed by conjugation and using the plasmid relaxase gene-typing method. All 37 isolates were multidrug resistant, and theblaKPC-2 gene was confirmed in all of them. In 17 isolates (45.9%), the blaCTX-M-2 gene was also amplified, as well as blaSHV-2 in five isolates (13.5%) and blaCTX-M-2/blaSHV-2 in four isolates (10.8%). The molecular epidemiology of the blaKPC-2 gene has resulted in it being associated with an IncL/M transferable plasmid disseminating in various sequence types (STs) (ST17, ST556, ST342, ST147, ST461, ST65, ST15 and ST70), and in a new genetic environment with a 764-bp deletion in the ISKpn7-blaKPC region. These findings contribute to the understanding of the great diversity of the blaKPC-2-carrying genetic platforms.

Dynamics of clonal and plasmid backgrounds of Enterobacteriaceae producing acquired AmpC in Portuguese clinical settings over time

The objective of this work was to provide detailed molecular data on clinically acquired AmpC (qAmpC)-producing Enterobacteriaceae from two different periods (2002-2008 and 2010-2013) in order to clarify the contribution of clonal and plasmid genetic platforms for the current epidemiological scenario concerning extended-spectrum beta-lactams resistance. We analysed 1246 Enterobacteriaceae non-susceptible to third-generation cephalosporins from two hospitals and one community laboratory between 2010 and 2013. Bacterial identification, antibiotic susceptibility, identification of qAmpC and plasmid-mediated quinolone resistance genes, clonal (pulsed-field gel electrophoresis (PFGE), Multilocus sequence typing (MLST)) and plasmid (S1-/I-CeuI-PFGE, replicon typing, hybridization) analysis were performed by standard methods. Whole-genome sequencing (WGS) was performed in two ST11-Klebsiella pneumoniae isolates harbouring DHA-1. The occurrence of qAmpC was lower (2.6%) than that observed in a previous survey (7.4%), and varied slightly over time. Isolates produced DHA-1 (53%), CMY-2 (44%) or DHA-6 (3%), but significant epidemiological changes were observed in the two surveys. While DHA-1 persisted in different institutions by selection of a worldwide epidemic IncR plasmid in an ST11 harbouring KL105, CMY-2 rates increased over time linked to IncI1 plasmids (instead of IncK or IncA/C2) in multiple Escherichia coli clones. The higher frequency of DHA-1 qAmpC in these species contrasts with the scenario in most European countries. Furthermore, the different genetic backgrounds associated with either extended-spectrum β-lactamases (ESBLs) or acquired AmpC β-lactamases (qAmpC) in our country might have contributed to their differential expansion.

Biocides and health-care agents are more than just antibiotics: Inducing cross to co-resistance in microbes

Health-care chemicals are used worldwide as important components of different industries as consumer products, food industry, animal husbandry and agribusiness. There are innumerable reports on the effect of these chemicals (biocides) impacting the development of cross to co-resistance in pathogenic bacteria. However, reports are limited on the concurrent use of agricides (pesticides, herbicides, fungicides and insecticides) which influence the microbial activities in soils and contribute to the increase in incidences of co-resistance. Undoubtedly, indiscriminate use of biocides and agricides has contaminated both water and soil environments. This review describes the onset of cross and co-resistance to biocides and antibiotics which is increasingly being exhibited by specific bacteria under a persistent selective pressure. It also re-examines the significance of mobile genetic platforms and horizontal gene transfer from one to another bacterial species, for understanding the kinetics and efficiency of genetic exchange in stressed environments leading to natural selection of tolerant strains over susceptible ones. The investigation is much warranted, particularly with respect to agricides that commonly occur in recalcitrant states in soil and water ecosystem, livestock, etc and is transmitted either directly or via the food-chain to human beings, facilitating the switch from cross to co-resistance.

Developing genetic manipulation platforms for Naegleria gruberi

Naegleria gruberi, is a free-living microbial eukaryote, which belongs to the group of Excavates. The organism is widely distributed especially in aquatic environments and it is famous for its ability to transform from an amoeba to flagellate and cyst forms depending on its surroundings. In silico examination of the published Naegleria gruberi genome opened up the possibility of functional exploration of the organism by molecular cell biology. Despite this, several attempts to genetically transfect or genetically manipulate the organism have been unsuccessful so far due to the unique morphological and cellular adaptations of the organisms, but also due to its resistance to certain basic antibiotics. Using a series of protocols and combination of cell biological tools, we attempted to genetic manipulate Naegleria using both CRISPR/Cas9 and traditional genetic transformation protocols. Preliminarily data from these investigations will be discussed. This work is going to provide traits found in the last eukaryotic common ancestor and provide a model for investigating the cell biology of other free-living eukaryotes.

Genome plasticity favours double chromosomal Tn4401b-blaKPC-2 transposon insertion in the Pseudomonas aeruginosa ST235 clone

BackgroundPseudomonas aeruginosa Sequence Type 235 is a clone that possesses an extraordinary ability to acquire mobile genetic elements and has been associated with the spread of resistance genes, including genes that encode for carbapenemases. Here, we aim to characterize the genetic platforms involved in resistance dissemination in blaKPC-2-positive P. aeruginosa ST235 in Colombia.ResultsIn a prospective surveillance study of infections in adult patients attended in five ICUs in five distant cities in Colombia, 58 isolates of P. aeruginosa were recovered, of which, 27 (46.6%) were resistant to carbapenems. The molecular analysis showed that 6 (22.2%) and 4 (14.8%) isolates harboured the blaVIM and blaKPC-2 genes, respectively. The four blaKPC-2-positive isolates showed a similar PFGE pulsotype and belonged to ST235. Complete genome sequencing of a representative ST235 isolate shows a unique chromosomal contig of 7097.241 bp with eight different resistance genes identified and five transposons: a Tn6162-like with ant(2″)-Ia, two Tn402-like with ant(3″)-Ia and blaOXA-2 and two Tn4401b with blaKPC-2. All transposons were inserted into the genomic islands. Interestingly, the two Tn4401b copies harbouring blaKPC-2 were adjacently inserted into a new genomic island (PAGI-17) with traces of a replicative transposition process. This double insertion was probably driven by several structural changes within the chromosomal region containing PAGI-17 in the ST235 background.ConclusionThis is the first report of a double Tn4401b chromosomal insertion in P. aeruginosa, just within a new genomic island (PAGI-17). This finding indicates once again the great genomic plasticity of this microorganism.

Emerging Molecular Technologies in Renal Cell Carcinoma: Liquid Biopsy.

Liquid biopsy, based on the circulating tumor cells (CTCs) and cell-free nucleic acids has potential applications at multiple points throughout the natural course of cancer, from diagnosis to follow-up. The advantages of doing ctDNA assessment vs. tissue-based genomic profile are the minimal procedural risk, the possibility to serial testing in order to monitor disease-relapse and response to therapy over time and to reduce hospitalization costs during the entire process. However, some critical issues related to ctDNA assays should be taken into consideration. The sensitivity of ctDNA assays depends on the assessment technique and genetic platforms used, on tumor-organ, stage, tumor heterogeneity, tumor clonality. The specificity is usually very high, whereas the concordance with tumor-based biopsy is generally low. In patients with renal cell carcinoma (RCC), qualitative analyses of ctDNA have been performed with interesting results regarding selective pressure from therapy, therapeutic resistance, exceptional treatment response to everolimus and mutations associated with aggressive behavior. Quantitative analyses showed variations of ccfDNA levels at different tumor stage. Compared to CTC assay, ctDNA is more stable than cells and easier to isolate. Splice variants, information at single-cell level and functional assays along with proteomics, transcriptomics and metabolomics studies can be performed only in CTCs.

The Spectrum of Genetic Testing.

To introduce genetic testing as it relates to oncology and nursing. Peer-reviewed journals, government web sites and resources, published recommendations, and professional experience as a genetic counselor. Genetic testing is a major component of oncology health care and with the continued expansion of the application of genetic testing, many patients will have genetic testing throughout their cancer journey. To provide supportive care for patients with cancer or at risk for cancer, oncology nurses need to appreciate the many and varied genetic testing platforms and testing strategies. Oncology nurses can be a resource for patients and family members regarding testing options, insurance coverage, and understanding medical management decisions.

Genetic platforms for heterologous expression of microbial natural products.

Covering: 2005 up to 2019Natural products are of paramount importance in human medicine. Not only are most antibacterial and anticancer drugs derived directly from or inspired by natural products, many other branches of medicine, such as immunology, neurology, and cardiology, have similarly benefited from natural product-based drugs. Typically, the genetic material required to synthesize a microbial specialized product is arranged in a multigene biosynthetic gene cluster (BGC), which codes for proteins associated with molecule construction, regulation, and transport. The ability to connect natural product compounds to BGCs and vice versa, along with ever-increasing knowledge of biosynthetic machineries, has spawned the field of genomics-guided natural product genome mining for the rational discovery of new chemical entities. One significant challenge in the field of natural product genome mining is how to rapidly link orphan biosynthetic genes to their associated chemical products. This review highlights state-of-the-art genetic platforms to identify, interrogate, and engineer BGCs from diverse microbial sources, which can be broken into three stages: (1) cloning and isolation of genomic loci, (2) heterologous expression in a host organism, and (3) genetic manipulation of cloned pathways. In the future, we envision natural product genome mining will be rapidly accelerated by de novo DNA synthesis and refactoring of whole biosynthetic pathways in combination with systematic heterologous expression methodologies.

Rebuilding Privacy Practices After Carpenter

The recent arrest of the alleged Golden State Killer has ignited law enforcement interest in using consumer genetic databases to crack cold cases. The break in that case came when investigators compared crime scene DNA to other DNA profiles searchable in an online genetic genealogy database called GEDmatch. Yet consumer genetic services have responded to law enforcement interest in markedly different ways. Some have explicitly denounced law enforcement use and vowed to oppose it; others have welcomed law enforcement expressly; and some have cooperated quietly with law enforcement, while keeping their users in the dark. At almost the same time, the Supreme Court gave these platforms a new role in policing police access to their genetic resources. In Carpenter v. United States, the Court upended the seemingly categorical rule that one cannot have an expectation of privacy in data shared with another. This Article examines the impact of Carpenter for law enforcement use of third-party DNA databases, as in the Golden State Killer case. In so doing, this Article makes three contributions. First, it joins a burgeoning scholarship in identifying Carpenter’s “test,” and demonstrates that genetic information is precisely the sort of data in which individuals may ordinarily maintain an expectation of privacy, even when that data is in third-party hands. Second, it considers the role of consumer genetic platforms in mediating police access to their resources, recasting third-party privacy practices in a more robust and nuanced role as measures of consent. Third, it assesses the privacy practices of genetic genealogy companies specifically, concluding that some plainly reinforce existing expectations of privacy in genetic data, while others have meandered their way closer to legally valid consent to government use—though none has done so with precision.

The C9 risk variant P167S and age-related macular degeneration

To evaluate genetic testing platforms used to aid in the diagnosis of inherited retinal degenerations (IRDs).Evaluation of diagnostic tests and technologies.Targeted genetic panel testing for IRDs.Data collected regarding targeted genetic panel testing for IRDs offered by different laboratories were investigated for the inclusion of coding and noncoding variants in disease genes. Both large IRD panels and smaller, more focused, disease-specific panels were included in the analysis.Number of disease genes tested as well as the commonality and uniqueness across testing platforms in both coding and noncoding variants of disease.Across the 3 IRD panel tests investigated, 409 unique genes are represented, of which 269 genes are tested by all 3 panels. The top 20 genes known to cause over 70% of all IRDs are represented in the 269 common genes tested by all 3 panels. In addition, 138 noncoding variants in 50 unique genes are assayed across the 3 platforms. Focused, disease-specific panels exhibit significant variability across the 5 testing platforms that were studied.Ordering genetic testing for IRDs is not straightforward, as evidenced by the multitude of panels available to providers. It is important that there is coverage of both coding and noncoding regions in IRD genes to offer diagnoses in these patients. This paper details the diversity of testing platforms currently available to clinicians and provides a thorough explanation of the genes tested in the different IRD panels. In a time of increased importance of the clinical genetic testing of patients with IRDs, knowledge of the proper test to order is paramount.

C-MET Overexpression as a Poor Predictor of MET Amplifications or Exon 14 Mutations in Lung Sarcomatoid Carcinomas

IntroductionMNNG HOS transforming gene (MET) abnormalities such as amplification and exon 14 mutations may be responsive to targeted therapies. They are prevalent in lung sarcomatoid carcinomas (LSCs) and must be diagnosed as efficiently as possible. Hypothetically, c-MET overexpression by immunohistochemistry (IHC) may prove effective as a screening test for MET abnormalities. MethodsTissue samples were obtained from consecutive patients with a resected LSC in four oncologic centers. IHC was performed using the SP44 antibody (Ventana, Tucson, Arizona) and evaluated using the MetMab score and H-score. Fluorescence in situ hybridization was applied with the dual color probe set from Zytovision (Clinisciences, Nanterre, France). True MET amplification was diagnosed when MET gene copy number was 5 or greater and the ratio between MET gene copy number and chromosome 7 number was greater than 2. All MET exon 14 alterations including those affecting splice sites occurring within splice donor and acceptor sites were detected in the routine molecular testing on genetic platforms. ResultsA total of 81 LSCs were included. Fourteen (17%) exhibited positive IHC using the MetMab score and 15 (18.5%) using the H-score. MET amplification was detected in six tumors (8.5%) and MET exon 14 mutation in five (6%). A weak positive correlation between IHC and fluorescence in situ hybridization was found (r = 0.27, p = 0.0001). IHC sensitivity for MET amplification was 50%, with a specificity of 83%, positive predictive value of 21.4%, and negative predictive value of 94.7%. IHC sensitivity for MET exon 14 mutations was 20%, with a specificity of 83%, positive predictive value of 7%, and negative predictive value of 94%. ConclusionIHC is not a relevant screening tool for MET abnormalities in LSC.

Characterisation of a class 1 integron associated with the formation of quadruple blaGES-5 cassettes from an IncP-1β group plasmid in Pseudomonas aeruginosa

Integrons are genetic platforms responsible for the dissemination of antimicrobial resistance genes among Gram-negative bacteria, primarily due to their association with transposable elements and conjugative plasmids. In this study, a cassette array containing four identical blaGES-5 genes embedded in a class 1 integron located on an IncP-1β group plasmid from a clinical Pseudomonas aeruginosa strain was identified. Comparative genome analysis and conjugation assay showed that the plasmid pICP-4GES lacked the trbN, trbO and trbP genes but was conjugable. Antimicrobial susceptibility testing revealed that compared with single-copy blaGES-5 complementary strains, both the cloned and chromosome-targeted expression of four copies of blaGES-5 increased the minimum inhibitory concentration (MIC) by one to two dilutions for most of the selected antimicrobials. Quantitative real-time reverse transcription PCR (RT-qPCR) showed that the four consecutive cassettes increased blaGES-5 expression by approximately two-fold compared with the single-copy blaGES-5 strain, suggesting that the level of gene expression was not directly proportional to copy number. In addition, the gene cassette capture assay showed that the global blaGES-5 transfer frequency reached 5.38 × 10-4.