Pulsed-field Electrophoresis Research Articles

Targeted phasing of 2–200 kilobase DNA fragments with a short-read sequencer and a single-tube linked-read library method

In the human genome, heterozygous sites refer to genomic positions with a different allele or nucleotide variant on the maternal and paternal chromosomes. Resolving these allelic differences by chromosomal copy, also known as phasing, is achievable on a short-read sequencer when using a library preparation method that captures long-range genomic information. TELL-Seq is a library preparation that captures long-range genomic information with the aid of molecular identifiers (barcodes). The same barcode is used to tag the reads derived from the same long DNA fragment within a range of up to 200 kilobases (kb), generating linked-reads. This strategy can be used to phase an entire genome. Here, we introduce a TELL-Seq protocol developed for targeted applications, enabling the phasing of enriched loci of varying sizes, purity levels, and heterozygosity. To validate this protocol, we phased 2–200 kb loci enriched with different methods: CRISPR/Cas9-mediated excision coupled with pulse-field electrophoresis for the longest fragments, CRISPR/Cas9-mediated protection from exonuclease digestion for mid-size fragments, and long PCR for the shortest fragments. All selected loci have known clinical relevance: BRCA1, BRCA2, MLH1, MSH2, MSH6, APC, PMS2, SCN5A-SCN10A, and PKI3CA. Collectively, the analyses show that TELL-Seq can accurately phase 2–200 kb targets using a short-read sequencer.

Prevalence and changing antimicrobial resistance profiles of Shigella spp. isolated from diarrheal patients in Kolkata during 2011-2019.

The primary aim of this study was to investigate the occurrence, characteristics, and antimicrobial resistance patterns of various Shigella serogroups isolated from patients with acute diarrhea of the Infectious Diseases Hospital in Kolkata from 2011-2019. During the study period, Shigella isolates were tested for their serogroups, antibiotic resistance pattern and virulence gene profiles. A total of 5.8% of Shigella spp. were isolated, among which S. flexneri (76.1%) was the highest, followed by S. sonnei (18.7%), S. boydii (3.4%), and S. dysenteriae (1.8%). Antimicrobial resistance against nalidixic acid was higher in almost all the Shigella isolates, while the resistance to β-lactamases, fluoroquinolones, tetracycline, and chloramphenicol diverged. The occurrence of multidrug resistance was found to be linked with various genes encoding drug-resistance, multiple mutations in the topoisomerase genes, and mobile genetic elements. All the isolates were positive for the invasion plasmid antigen H gene (ipaH). Dendrogram analysis of the plasmid and pulsed-field electrophoresis (PFGE) profiles revealed 70-80% clonal similarity among each Shigella serotype. This comprehensive long-term surveillance report highlights the clonal diversity of clinical Shigella strains circulating in Kolkata, India, and shows alarming resistance trends towards recommended antibiotics. The elucidation of this study's outcome is helpful not only in identifying emerging antimicrobial resistance patterns of Shigella spp. but also in developing treatment guidelines appropriate for this region.

Feasibility of Optical Genome Mapping from Placental and Umbilical Cord Sampled after Spontaneous or Therapeutic Pregnancy Termination

Optical genome mapping (OGM) is an alternative to classical cytogenetic techniques to improve the detection rate of clinically significant genomic abnormalities. The isolation of high-molecular-weight (HMW) DNA is critical for a successful OGM analysis. HMW DNA quality depends on tissue type, sample size, and storage conditions. We assessed the feasibility of OGM analysis of DNA from nine umbilical cord (UC) and six chorionic villus (CV) samples collected after the spontaneous or therapeutic termination of pregnancy. We analyzed quality control metrics provided by the Saphyr system (Bionano Genomics) and assessed the length of extracted DNA molecules using pulsed-field capillary electrophoresis. OMG data were successfully analyzed for all six CV samples. Five of the UC samples did not meet the Saphyr quality criteria, mainly due to poor DNA quality. In this regard, we found that DNA quality assessment with pulsed-field capillary electrophoresis can predict a successful OGM analysis. OGM data were fully concordant with the results of standard cytogenetic methods. Moreover, OGM detected an average of 14 additional structural variants involving OMIM genes per sample. On the basis of our results, we established the optimal conditions for sample storage and preparation required for a successful OGM analysis. We recommend checking DNA quality before analysis with pulsed-field capillary electrophoresis if the storage conditions were not ideal or if the quality of the sample is poor. OGM can therefore be performed on fetal tissue harvested after the termination of pregnancy, which opens up the perspective for improved diagnostic yield.

Increasing of New CA-MRSA Infections Detected in people living with HIV Who Engage in Chemsex in Barcelona: An Ambispective Study.

There are no data on community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections in the context of the chemsex phenomenon. This study aimed to characterize CA-MRSA-related infections in a cohort of people living with HIV (PLWH) who engage in chemsex. At the Hospital Clinic of Barcelona, from February 2018 to January 2022, we analyzed CA-MRSA infections diagnosed in a cohort of PLWH who engage in chemsex. Epidemiological, behavioral and clinical variables were assessed. Mass spectrometry identification and antimicrobial susceptibility testing were performed on MRSA isolates. Pulse field electrophoresis was used to assess the clonality of the MRSA strains. The presence of Panton-Valentine leukocidin was also investigated. Among the cohort of 299 participants who engage in chemsex, 25 (8%) with CA-MRSA infections were identified, 9 at baseline and 16 with incident cases; the cumulative incidence was 5.5% (95% CI: 3.2%, 8.8%). The most common drugs were methamphetamine (96%) and GHB/GBL (92%). Poly-consumption and slamming were reported by 32% and 46%, respectively. CA-MRSA was isolated from the infection sites of 20 participants, and CA-MRSA colonization was confirmed in the remaining 5 persons. Seventy-one percent had used antibiotics in the previous year. All participants presented with skin and soft tissue infections, 28% required hospitalization, and 48% had recurrence. Of the 23 MRSA isolates further studied, 19 (82,6%) belonged to the same clone. Panton-Valentine leukocidin was detected in all isolates. PLWH who engage in chemsex may present with CA-MRSA infections. Clinical suspicion and microbiological diagnosis are required to provide adequate therapy, and CA-MRSA prevention interventions should be designed.

One Health Approach: Invasive California Kingsnake (Lampropeltis californiae) as an Important Source of Antimicrobial Drug-Resistant Salmonella Clones on Gran Canaria Island.

The increase in the reptile population has led to a rise in the number of zoonotic infections due to close contact with reptiles, with reptile-associated salmonellosis being particularly relevant. California kingsnake invasion not only threatens the endemic reptile population of the island of Gran Canaria (Spain) but also poses serious public health problems by spreading zoonotic pathogens and their antimicrobial resistance (AMR) to the environment. Thus, the aim of this study was to assess the occurrence, genetic diversity, and AMR among Salmonella spp. strains isolated from California kingsnakes in Gran Canaria Island (Spain). Of 73 invasive individuals captured, 20.5% carried Salmonella spp., belonging to different subspecies and serovars, with subsp. salamae as the most abundant. Pulsed-field electrophoresis showed high genetic diversity among subsp. salamae isolates, and among these, 73.3% showed resistance to at least one of the antimicrobials tested. In conclusion, the present study revealed the importance of wild invasive California kingsnakes as reservoirs of drug-resistant Salmonella spp. that could pose a direct threat to livestock and humans. Identification of drug-resistant Salmonella strains in wildlife provides valuable information on potential routes of transmission that involve risks to public and animal health.

Evaluation of core genome and whole genome multilocus sequence typing schemes for Campylobacter jejuni and Campylobacter coli outbreak detection in the USA.

Campylobacter is a leading causing of bacterial foodborne and zoonotic illnesses in the USA. Pulsed-field gene electrophoresis (PFGE) and 7-gene multilocus sequence typing (MLST) have been historically used to differentiate sporadic from outbreak Campylobacter isolates. Whole genome sequencing (WGS) has been shown to provide superior resolution and concordance with epidemiological data when compared with PFGE and 7-gene MLST during outbreak investigations. In this study, we evaluated epidemiological concordance for high-quality SNP (hqSNP), core genome (cg)MLST and whole genome (wg)MLST to cluster or differentiate outbreak-associated and sporadic Campylobacter jejuni and Campylobacter coli isolates. Phylogenetic hqSNP, cgMLST and wgMLST analyses were also compared using Baker's gamma index (BGI) and cophenetic correlation coefficients. Pairwise distances comparing all three analysis methods were compared using linear regression models. Our results showed that 68/73 sporadic C. jejuni and C. coli isolates were differentiated from outbreak-associated isolates using all three methods. There was a high correlation between cgMLST and wgMLST analyses of the isolates; the BGI, cophenetic correlation coefficient, linear regression model R 2 and Pearson correlation coefficients were >0.90. The correlation was sometimes lower comparing hqSNP analysis to the MLST-based methods; the linear regression model R 2 and Pearson correlation coefficients were between 0.60 and 0.86, and the BGI and cophenetic correlation coefficient were between 0.63 and 0.86 for some outbreak isolates. We demonstrated that C. jejuni and C. coli isolates clustered in concordance with epidemiological data using WGS-based analysis methods. Discrepancies between allele and SNP-based approaches may reflect the differences between how genomic variation (SNPs and indels) are captured between the two methods. Since cgMLST examines allele differences in genes that are common in most isolates being compared, it is well suited to surveillance: searching large genomic databases for similar isolates is easily and efficiently done using allelic profiles. On the other hand, use of an hqSNP approach is much more computer intensive and not scalable to large sets of genomes. If further resolution between potential outbreak isolates is needed, wgMLST or hqSNP analysis can be used.

Automated phenol-chloroform extraction of high molecular weight genomic DNA for use in long-read single-molecule sequencing.

Background: Automation has increasingly become more commonplace in the research laboratory workspace. The introduction of articulated robotic arms allows the researcher more flexibility in the tasks a single piece of automated machinery can perform. We set out to incorporate automation in processing of genomic DNA organic extractions to increase throughput and limit researchers to the exposure of organic solvents. Methods: In order to automate the genome sequencing pipeline in our laboratory, we programmed a dual-arm anthropomorphic robot, the Robotic Biology Institute's Maholo LabDroid, to perform organic solvent-based genomic DNA extraction from cell lysates. To the best of our knowledge, this is the first time that automation of phenol-chloroform extraction has been reported. Results: We achieved routine extraction of high molecular weight genomic DNA (>100 kb) from diverse biological samples including algae cultured in sea water, bacteria, whole insects, and human cell lines. The results of pulse-field electrophoresis size analysis and the N50 sequencing metrics of reads obtained from Nanopore MinION runs verified the presence of intact DNA suitable for direct sequencing. Conclusions: We present the workflow that can be used to program similar robots and discuss the problems and solutions we encountered in developing the workflow. The protocol can be adapted to analogous methods such as RNA extraction, and there is ongoing work to incorporate further post-extraction steps such as library construction. This work shows the potential for automated robotic workflows to free molecular biological researchers from manual interventions in routine experimental work. A time-lapse movie of the entire automated run is included in this report.

Separation of proteins by square-wave pulsed field and inversion field capillary electrophoresis

BackgroundCapillary electrophoresis (CE) is an effective method for protein analysis. Although previous researchers have proved that the separation performance of large DNA molecules was improved by pulsed field capillary electrophoresis (PFCE), there is lack of research about proteins separation by PFCE. MethodsHerein, we systematically investigated the separation performance of proteins sized from 44 kDa to 200 kDa under square wave and inversion electric field, respectively. FindingsResults showed that for pulsed electric field with same forward and backward time duration, there was a resonance frequency for protein molecule re-orientation and applied frequency, and the apparent mobility (μ) was the lowest at the resonance frequency. μ was positively correlated to modulation depth (M) if M was below 200%, while μ was negatively correlated to M if M was higher than 200%. For CE with same forward and backward voltage, it demonstrated that μ dropped as M went up, but the separation performance for proteins with high molecular mass improved. Through PFCE, albumin and α- globulins in human serum were also resolved within 7 min. Such a work may shed new light on the analysis of proteins by PFCE.

Molecular characterization of A. baumanni isolates causing co-infections in SARS-COV-2 patients

Abstract Background Respiratory viral infections, such as COVID-19, predispose patients to co-infections leading to increased morbidity and mortality. A. baumannii poses as a serious threat to hospital facilities because of its ability to persist in the environment and acquire multi-drug resistance. The aim of this study was to quantify the extent of A. baumannii cross-infection and identify any gene clonality between isolates in SARS-CoV-2 patients. Methods Bacterial isolates of A. baumannii found in patients with SARS-CoV-2 admitted to the main Intensive Care Unit (ICU) of the Umberto I Teaching Hospital of Rome were collected between March 2020 and February 2021. Isolates were typed by pulsed-field electrophoresis to analyse their homology relationships. Results Overall, 196 SARS-CoV-2 patients were admitted to the ICU. They were mainly male (N = 138) and aged 63 years on average. Of these, 122 died, and 74 were discharged. A total of 157 strains of A. baumannii were isolated from 74 patients (38%), who had a higher mean hospital stay than patients in whom the bacterial strain had not been isolated (24.6 vs. 12.2 days). The genotypic analysis of 120 isolates revealed two main patterns (A and F) and a few subtypes, especially A8 (43%), A4 (29%), and A11 (10%). Clone A8 was found mainly between October 2020 and February 2021, clone A4 in April-December 2020 and January-February 2021, and A11 in December 2020 and January 2021. The strains were susceptible to colistin only, were isolated mostly from tracheobronchial aspirates (41%) or rectal swabs (35%) and accounted for 56 healthcare-associated infections (33% of which sustained by A4, 38% by A8, and 9% by A11). Conclusions The isolation of A. baumannii from patients with COVID-19 highlighted the importance of monitoring co-infections caused by this pathogen, which frequently shows a multi-drug resistant profile that may lengthen the hospital stay. It is essential to implement preventive measures to contain these infections. Key messages A. baumanni is a pathogen that needs to be monitored because it may lengthen the hospital stay of SARS-CoV-2 patients. In critically ill patients, the continued growth of multidrug-resistant organisms shows the importance of preventing these infections.

Infections in patients colonized with carbapenem-resistant Gram-negative bacteria in a medium Spanish city

RESUMENObjetivoDebido a que existen pocos estudios sobre las implicaciones clínicas de la colonización por bacterias gramnegativas resistentes a carbapenémicos (BRC) se analizó ésta en frotis rectales (FR) y faríngeos (FF) y su relación con la capacidad de predecir infección/colonización.Material y métodosSe realizó un estudio transversal, retrospectivo de los pacientes adultos hospitalizados entre enero del 2016 y diciembre del 2019. Los aislamientos fueron caracterizados mediante MicroScan y espectrometría de masas, aplicando los puntos de corte EUCAST 2018. La detección de carbapenemasas se realizó mediante PCR y secuenciación Sanger; se asignó el secuenciotipo mediante MLST. La relación genética entre los aislados se hizo mediante electroforesis de campo pulsado usando las enzimas Xbal, Spel o Apal.Resultados. Se detectaron 308 (86,03 %) FR y 50 (13,97%) FF positivos, teniendo el FR una sensibilidad del 85%, especificidad del 100%, VPP 100% y VPN 97%. En los FR se aislaron: 44% (n=135) Acinetobacter baumannii, 26% (n=80) Enterobacterales (20 KPC, 29 OXA-48, 22 VIM, 2 IMP, 7 NDM), 17% (n=53) Pseudomonas aeruginosa y 13% (n=40) Stenotrophomonas maltophilia. En los FF se aislaron un 44% (n=22) S. maltophilia, 40% (n=20) A. baumannii, 8% (n=4) P. aeruginosa y 8% (n=4) Enterobacterales (3 VIM, 1 OXA). De los pacientes con tomas simultáneas de FR y FF, 41 (40,6%) tuvieron positividad en ambos frotis, 45 (44,6%) sólo en FR y 15 (14,9%) sólo en FF. En el 81,3% (n=13) de los episodios la colonización precedió a la infección, existiendo asociación entre infección y colonización (p<0,001; χ2) y en todos en los que se conservó la información del pulsotipo los aislados de las muestras clínicas y de los frotis fueron similares.ConclusionesLa probabilidad de predecir la infección a través del colonizado por BRC en diferentes muestras clínicas es factible, teniendo el FR una mayor sensibilidad para detectar colonización.

E. coli O157:H7 outbreak and hemolytic uremic syndrome in a day care center in Brazil

Background: Shiga toxin-producing Escherichia coli O157:H7 (STEC-O157: H7) infection is related to major outbreaks and serious diseases such as hemolytic uremic syndrome (HUS). In March 2019, a gastroenteritis outbreak occurred in the day care center of Vila Velha-Brazil, and a death occurred by HUS. Epidemiological investigation was conducted to identify the agent involved and source of infection. Methods and materials: A descriptive study. Cases were: children or employees who presented diarrhea or vomiting from March 1st to April 5th, 2019. A semi-structured questionnaire was applied. The rate attack of cases per class was calculated. Stool samples were collected from the cases and from water reservoir, kitchen tap reducer, water toy, water and food. Laboratory techniques used: culture, Polymerase chain reaction (PCR), and pulsed field electrophoresis- PFGE. Results: There were 24 cases of diarrhea, of these two (79,0%) were children, five (20,9%) employees. The cases reduced by 92% after the suspension of day care activities in March 26th. The 2-year-old class had the highest attack rate (40,0%) and there were no case in the 4-year-old class. Bloody diarrhea was reported in five (21,0%), abdominal pain nine (38,0%), vomiting eight (33,0%), and three (12,0%) evolved to HUS, with one death (33% lethality by SHU). STEC-O157: H7 was detected in two children who had bloody diarrhea (one with HUS) and in the water tap reducer. The PFGE analysis identified that, the genetic homology between case strains was 90% and the genetic similarity between the cases and the water tap reducer was 64,0%. Conclusion: A gastroenteritis outbreak occurred in the day care center involving the EHEC O157: H7 strain. It is suggested that E. coli was introduced into the day care center by an infected person, spreading the outbreak, maintained by person-to-person transmission, stop the school activities and professionals guide about the characteristics of the disease helped in minimizing the severity of cases. It was recommended to guide teachers/staff about how to prevent fecal-oral transmission diseases, and to monitor water quality in the day care center.

Prevalence of carbapenem-resistant Enterobacteriaceae and emergence of high rectal colonization rates of blaOXA-181-positive isolates in patients admitted to two major hospital intensive care units in Kuwait.

BackgroundFecal colonization by carbapenem-resistant Enterobacteriaceae (CRE) can be the main reservoir for transmission of these resistant organisms especially in the Intensive Care Units (ICUs).AimThis study was conducted to evaluate the rate of rectal carriage and molecular characterization of CRE in patients hospitalized in the ICUs of 2 major hospitals (Adan and Mubarak Al Kabeer Hospitals) in Kuwait.Materials and methodsRectal swabs were collected from all patients at admission, 48 h after admission and once weekly from April 2017- March 2018. Initial CRE screening was carried out on MacConkey agar on which meropenem disc 10μg was placed. Identification of isolates was by API 20E. Susceptibility testing was performed using the E-test method. Polymerase chain reaction (PCR) was used to detect the carbapenemase-encoding genes. Clonal relationship was investigated by pulsed-field electrophoresis (PFGE). Genes of blaOXA-181 and blaNDM-5–carrying plasmids were detected in some strains.ResultsA total of 590 patients were recruited into the study. Of these, 58 were positive for CRE, giving a prevalence of 9.8%; 25/320 (7.8%) in Adan and 33/270 (12.2%) in Mubarak Al Kabeer Hospitals. All isolates were resistant to multiple antibiotics. Resistance rates to colistin and tigecycline were 17% and 83%, respectively. Single genes of blaOXA-181 were detected in isolates from 38 (65.5%) out of 58 patients and in 5 patients colonized by blaOXA-48-positive CRE. A combination of 2 genes was detected in 12 isolates; 5 blaKPC-2 and blaOXA-181, 4 blaVIM-1 and blaOXA-181, and 3 blaNDM-5 and blaOXA-181. PFGE showed an overall level of similarity of 38%. Southern hybridization studies localized the blaOXA-181 and blaNDM-5 genes to a large plasmid of 200kb in 3 K. pneumoniae isolates and a small plasmid of 80kb in 2 E. coli isolates, respectively.ConclusionThe prevalence of CRE colonization in the 2 hospital ICUs was relatively high and the emergence of blaOXA-181-mediated CRE is a cause for concern as there is the possibility of rapid horizontal spread among hospital patients in Kuwait. Active surveillance of CRE in the ICUs is highly recommended to stem its spread.

Clonal outbreak caused by VIM-4-producing Proteus mirabilis in a Greek tertiary-care hospital

Carbapenem-resistant Enterobacterales have become a major public-health issue worldwide. Here we report an outbreak caused by a clonal multidrug-resistant Proteus mirabilis strain producing VIM-4 metallo-β-lactamase (MBL) and TEM-2 β-lactamase in a Greek tertiary-care hospital. From July 2015 to February 2016, 27 imipenem-resistant P. mirabilis isolates were recovered from 14 patients hospitalised in two intensive care units (ICUs) and the internal medicine department in AHEPA University Hospital, Thessaloniki. The isolates were either susceptible or resistant to meropenem and were resistant to all remaining β-lactams except aztreonam. Phenotypic and molecular analysis revealed that all of the isolates harboured a blaVIM-4 MBL gene. Resistome analysis of a representative isolate showed the presence of an IncQ1 plasmid harbouring the blaVIM-4 carbapenemase and blaTEM-2 β-lactamase genes among resistance genes coding for resistance to β-lactams, aminoglycosides, trimethoprim, sulfonamides and lincosamides. Genotyping by pulsed-field electrophoresis (PFGE) revealed that the isolates were epidemiologically related. After recovery of the index carbapenemase-producing P. mirabilis clinical isolate, infection control measures were intensified in the affected departments. Rectal sampling for carbapenem-resistant bacteria was initiated on a weekly basis among patients admitted to the general ICU. The outbreak was finally interrupted 6 months later in February 2016. This is the first documentation of the blaVIM-4 MBL gene in P. mirabilis as well as the first hospital outbreak caused by a MBL-producing P. mirabilis strain.

Pulse-Field capillary electrophoresis of repeat-primed PCR amplicons for analysis of large repeats in Spinocerebellar Ataxia Type 10.

Large expansions of microsatellite DNA cause several neurological diseases. In Spinocerebellar ataxia type 10 (SCA10), the repeat interruptions change disease phenotype; an (ATTCC)n or a (ATCCT)n/(ATCCC)n interruption within the (ATTCT)n repeat is associated with the robust phenotype of ataxia and epilepsy while mostly pure (ATTCT)n may have reduced penetrance. Large repeat expansions of SCA10, and many other microsatellite expansions, can exceed 10,000 base pairs (bp) in size. Conventional next generation sequencing (NGS) technologies are ineffective in determining internal sequence contents or size of these expanded repeats. Using repeat primed PCR (RP-PCR) in conjunction with a high-sensitivity pulsed-field capillary electrophoresis fragment analyzer (FEMTO-Pulse, Agilent, Santa Clara, CA) (RP-FEMTO hereafter), we successfully determined sequence content of large expansion repeats in genomic DNA of SCA10 patients and transformed yeast artificial chromosomes containing SCA10 repeats. This RP-FEMTO is a simple and economical methodology which could complement emerging NGS for very long sequence reads such as Single Molecule, Real-Time (SMRT) and nanopore sequencing technologies.

Chloroplast nucleoids are highly dynamic in ploidy, number, and structure during angiosperm leaf development.

Chloroplast nucleoids are large, compact nucleoprotein structures containing multiple copies of the plastid genome. Studies on structural and quantitative changes of plastid DNA (ptDNA) during leaf development are scarce and have produced controversial data. We have systematically investigated nucleoid dynamics and ptDNA quantities in the mesophyll of Arabidopsis, tobacco, sugar beet, and maize from the early post-meristematic stage until necrosis. DNA of individual nucleoids was quantified by DAPI-based supersensitive epifluorescence microscopy. Nucleoids occurred in scattered, stacked, or ring-shaped arrangements and in recurring patterns during leaf development that was remarkably similar between the species studied. Nucleoids per organelle varied from a few in meristematic plastids to >30 in mature chloroplasts (corresponding to about 20-750 nucleoids per cell). Nucleoid ploidies ranged from haploid to >20-fold even within individual organelles, with average values between 2.6-fold and 6.7-fold and little changes during leaf development. DNA quantities per organelle increased gradually from about a dozen plastome copies in tiny plastids of apex cells to 70-130 copies in chloroplasts of about 7μm diameter in mature mesophyll tissue, and from about 80 plastome copies in meristematic cells to 2600-3300 copies in mature diploid mesophyll cells without conspicuous decline during leaf development. Pulsed-field electrophoresis, restriction of high-molecular-weight DNA from chloroplasts and gerontoplasts, and CsCl equilibrium centrifugation of single-stranded and double-stranded ptDNA revealed no noticeable fragmentation of the organelle DNA during leaf development, implying that plastid genomes in mesophyll tissues are remarkably stable until senescence.

Tracking KPC-3-producing ST-258 Klebsiella pneumoniae outbreak in a third-level hospital in Granada (Andalusia, Spain) by risk factors and molecular characteristics.

The objective of this study was to determine clinical-epidemiological characteristics of the patients and the genetic characteristics of carbapenemase KPC-3-producing Klebsiella pneumoniae isolates belonging to sequence type ST258. The eligible study population was all patients with isolates detected between October 2015 and March 2017. Clinical-epidemiological and microbiological data were gathered on risk factors associated with infection by this clone. Antimicrobial susceptibility was determined using MicroScan system and diffusion in agar. Genes encoding carbapenemases were detected using PCR and Sanger sequencing. The sequence type was assigned by MLST, and the genetic relationship among clinical isolates was determined by pulsed field electrophoresis and by analysis of the genetic environment. The study included 23 individuals with isolates of KPC-3/ST258; the mean age was 77year, and mean stay pre-isolation was 32days; 81% received empirical antimicrobial treatment. Isolates were only susceptible to gentamicin (CIM ≤ 2mg/L), tigecycline (CIM ≤ 1mg/L), and colistin (CIM ≤ 2mg/L). The isolates belonged to ST258, with five pulse types or subgroups. All isolates showed amplification of KPC, which was identified as KPC-3 variant. Gene blaKPC-3 was flanked by insertion sequences Kpn6 and Kpn7 within Tn4401 transposon isoform a. We report, for the first time in Spain, an 18-month outbreak by KPC-3-producing ST258 K. pneumoniae. Its acquisition was associated with a history of antimicrobial therapy, with three treatment options, and with high mortality. The detection of different pulse types is attributable to different introductions of the clone in our setting, supporting the need for multi-resistant isolate surveillance studies.

Cibler les recommandations d'hygiène sur la base de résultats microbiologiques précis

Tap water Pseudomonas aeruginosa colonization in the intensive care unit of the hospital, invites us to measure the extent of this contamination by multiplying the samples. In parallel, hospitalised patients which should be colonized or infected by the same microorganism were prospectively detected..Over a period of four months, isolate in nine patients strains of P. aeruginosa will be compared by pulsed-field electrophoresis with environmental strains to better understand the origin of contaminations and assess the risk of cross-transmission during nursing and cares.Finally a strain of P. aeruginosa isolated from the water delivered by the tap of one of the rooms successively contaminates the patients hospitalised in this room. The genotypic comparisons of the strains confirm the absence of cross-transmission within the service, each colonized or infected patient obviously has it from its endogenous flora.These results provided the necessary arguments for the profound change in the service’s water network architecture to simplify it and simplify maintenance. They reinforce the hygiene team and reassure service providers about the quality of care they provide.

The impact of active dry yeasts in commercial wineries from the Denomination of Origen "Vinos de Madrid", Spain.

This paper has studied the success of implantation for 16 commercial active dry yeasts (ADYs) during industrial fermentation (30) and the impact of these yeasts during spontaneous fermentations (19) in 10 wineries from the Denomination of Origin "Vinos de Madrid" over two consecutive years. Yeasts strains were identified by molecular techniques, pulsed field electrophoresis and microsatellite analysis. According to these techniques, all the ADYs were different with the exceptions of two strains, L2056 and Rh, which showed the same karyotype and loci size. The results showed that inoculating fermentations with ADYs did not ensure their dominance throughout the fermentation; the implantation level of ADYs was above 80% in only 9 of the 30 commercial fermentations studied; while in 16 fermentations, the dominance of the inoculated ADYs was below 50%. The type of vinification with the best implantation results overall were those associated with red wine fermentations. ADYs affected spontaneous fermentations, although their impact was observed to decrease in the second year of the study. Therefore, specific adaptation studies are necessary before using commercial yeasts during the fermentation process. At the same time, a study was carried out on the frequency of commercial strains in IMIDRA's yeast collection, made up of strains isolated from spontaneous fermentations of the different areas and cellars since the beginning of the Denomination of Origin "Vinos de Madrid" in 1990. Six different ADYs were found with a frequency of less than 5%.

Pulsed-Field Gel Electrophoresis Typing of Staphylococcus aureus Strains.

Pulsed-field gel electrophoresis (PFGE) is one of the most effective genotyping methods for the study of genetic relatedness of microorganisms as well as for epidemiologic investigation of outbreaks caused by pathogenic bacteria, including Staphylococcus aureus. The technique relies on the size patterns of large DNA fragments generated by the cleavage of intact bacterial chromosomes with a rare cutting restriction enzyme, and subsequently resolved by pulsed-field electrophoresis with periodic changes of the orientation of the electrical field across the gel. The high discriminatory power, improved reproducibility by standardization of experimental protocols and data analysis guidelines, and establishment of a national PFGE database of S. aureus profiles have made it a valuable means for global tracking of S. aureus infection sources and determination of genetic relatedness of the strains.

Rewiring E2F1 with classical NHEJ via APLF suppression promotes bladder cancer invasiveness

BackgroundBladder cancer progression has been associated with dysfunctional repair of double-strand breaks (DSB), a deleterious type of DNA lesions that fuel genomic instability. Accurate DSB repair relies on two distinct pathways, homologous recombination (HR) and classical non-homologous end-joining (c-NHEJ). The transcription factor E2F1 supports HR-mediated DSB repair and protects genomic stability. However, invasive bladder cancers (BC) display, in contrast to non-invasive stages, genomic instability despite their high E2F1 levels. Hence, E2F1 is either inefficient in controlling DSB repair in this setting, or rewires the repair apparatus towards alternative, error-prone DSB processing pathways.MethodsRT-PCR and immunoblotting, in combination with bioinformatics tools were applied to monitor c-NHEJ factors status in high-E2F1-expressing, invasive BC versus low-E2F1-expressing, non-invasive BC. In vivo binding of E2F1 on target gene promoters was demonstrated by ChIP assays and E2F1 CRISPR-Cas9 knockdown. MIR888-dependent inhibition of APLF by E2F1 was demonstrated using overexpression and knockdown experiments, in combination with luciferase assays. Methylation status of MIR888 promoter was monitored by methylation-specific PCR. The changes in invasion potential and the DSB repair efficiency were estimated by Boyden chamber assays and pulse field electrophoresis, correspondingly.ResultsHerein, we show that E2F1 directly transactivates the c-NHEJ core factors Artemis, DNA-PKcs, ligase IV, NHEJ1, Ku70/Ku80 and XRCC4, but indirectly inhibits APLF, a chromatin modifier regulating c-NHEJ. Inhibition is achieved by miR-888-5p, a testis-specific, X-linked miRNA which, in normal tissues, is often silenced via promoter methylation. Upon hypomethylation in invasive BC cells, MIR888 is transactivated by E2F1 and represses APLF. Consequently, E2F1/miR-888/APLF rewiring is established, generating conditions of APLF scarcity that compromise proper c-NHEJ function. Perturbation of the E2F1/miR-888/APLF axis restores c-NHEJ and ameliorates cell invasiveness. Depletion of miR-888 can establish a ‘high E2F1/APLF/DCLRE1C’ signature, which was found to be particularly favorable for BC patient survival.ConclusionSuppression of the ‘out-of-context’ activity of miR-888 improves DSB repair and impedes invasiveness by restoring APLF.