Analysis of Vancomycin-Resistant Enterococci in Hemato-Oncological Patients

Enterococci are important bacterial pathogens, and their significance is even greater in the case of vancomycin-resistant enterococci (VRE). The study analyzed the presence of VRE in the gastrointestinal tract (GIT) of hemato-oncological patients. Active screening using selective agars yielded VRE for phenotypic and genotypic analyses. Isolated strains were identified with MALDI-TOF MS, (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry) their susceptibility to antibiotics was tested, and resistance genes (vanA, vanB, vanC-1, vanC2-C3) and genes encoding virulence factors (asa1, gelE, cylA, esp, hyl) were detected. Pulsed-field gel electrophoresis was used to assess the relationship of the isolated strains. Over a period of three years, 103 VanA-type VRE were identified in 1405 hemato-oncological patients. The most frequently detected virulence factor was extracellular surface protein (84%), followed by hyaluronidase (40%). Unique restriction profiles were observed in 33% of strains; clonality was detected in 67% of isolates. The study found that 7% of hemato-oncological patients carried VRE in their GIT. In all cases, the species identified was Enterococcus faecium. No clone persisted for the entire 3-year study period. However, genetically different clusters were observed for shorter periods of time, no longer than eight months, with identical VRE spreading among patients.

Methylase-assisted subcloning for high throughput BioBrick assembly

The BioBrick standard makes possible iterated pairwise assembly of cloned parts without any depletion of unique restriction sites. Every part that conforms to the standard is compatible with every other part, thereby fostering a worldwide user community. The assembly methods, however, are labor intensive or inefficient compared to some newer ones so the standard may be falling out of favor. An easier way to assemble BioBricks is described herein. Plasmids encoding BioBrick parts are purified from Escherichia coli cells that express a foreign site-specific DNA methyltransferase, so that each is subsequently protected in vitro from the activity of a particular restriction endonuclease. Each plasmid is double-digested and all resulting restriction fragments are ligated together without gel purification. The ligation products are subsequently double-digested with another pair of restriction endonucleases so only the desired insert-recipient vector construct retains the capacity to transform E. coli. This 4R/2M BioBrick assembly protocol is more efficient and accurate than established workflows including 3A assembly. It is also much easier than gel purification to miniaturize, automate and perform more assembly reactions in parallel. As such, it should streamline DNA assembly for the existing community of BioBrick users, and possibly encourage others to join.

Specific Detection and Identification of Fusarium graminearum Sensu Stricto Using a PCR-RFLP Tool and Specific Primers Targeting the Translational Elongation Factor 1α Gene

Fusarium graminearum is a toxigenic plant pathogen that causes Fusarium head blight (FHB) disease on cereal crops. It has recently shown to have cross-pathogenicity on noncereals (i.e., Fusarium root rot [FRR] on soybean) in Canada and elsewhere. Specific detection and differentiation of this potent toxigenic, trichothecene-producing pathogen among other closely related species is extremely important for disease control and mycotoxin monitoring. Here, we designed a PCR restriction fragment length polymorphism protocol based on the DNA sequence of the translational elongation factor 1α (TEF1α) gene. A unique restriction site to the enzyme HpaII is only found in F. graminearum sensu stricto strains among different Fusarium strains in the F. graminearum species complex (FGSC) and other Fusarium spp. associated with FHB in cereals and FRR in soybean. Partial amplification of the TEF1α gene with newly designed primers mh1/mh2 generated a 459-bp PCR fragment. Restriction digestion of the generated fragments with the HpaII enzyme generated a unique restriction pattern that can rapidly and accurately differentiate F. graminearum sensu stricto among all other Fusarium spp. A primer pair (FgssF/FgssR) specific to F. graminearum sensu stricto also was designed and can distinguish F. graminearum sensu stricto from all other Fusarium spp. in the FGSC and other closely related Fusarium spp. involved in FHB and FRR. This finding will be very useful for the specific detection of F. graminearum sensu stricto for diagnostic purposes as well as for the accurate detection of this pathogen in breeding and other research purposes.

Identification of Cladosporium sp. Fungi by in- silico RFLP-PCR

Cladosporium sp. plays an important role in human health, it is one of the pathogenic fungi which cause allergy and asthma and most frequently isolated from airborne spores. In this study, a couple of universal PCR primers were designed to identify the pathogenic fungi Cladosporium sp. according to conserved region 5.8S, 18S and 28S subunit ribosomal RNA gene in Cladosporium species. In silico RFLP-PCR were used to identify twenty-four Cladosporium strains. The results showed that the universal primer has the specificity to amplify the conserved region in 24 species as a band in virtual agarose gel. They also showed that the RFLP method is able to identify three Cladosporium species by specific and unique restriction enzymes for each one. These species are Cl. halotorenas by the two unique enzymes BsaXI and MobII, the other species is Cl. colrandse by two enzymes BccI and BtsCI, while the third species is Cl. aciculare by one enzyme BceAI. Each enzyme forms two bands in virtual agarose gel as a results of cutting the DNA by the enzyme, where the rest twenty – two species share more than one restriction enzymes. This method is active and rapid for identifying Cladosporium genus and three species by computational bases methods before applying it in the lab for more accuracy, efficiency, and specificity of designed primer to get good results in a short time.

Evidence for Infections by the Same Strain of Beta 2-toxigenic Clostridium perfringens Type A Acquired in One Hospital Ward

This study conducts a comparative phenotypic and genetic analysis of C. perfringens strains isolated from two patients hospitalized at the same time in 2017 in the surgical ward of the Provincial Specialist Hospital in Włocławek (Kujawsko-Pomorskie Province) who developed necrotizing soft tissue infections (NSTI). To explain the recurring cases of this infection, a comparative analysis was performed for these strains and the ones originating from infections recorded at the same hospital in three patients with gas gangrene in 2015. The two C. perfringens isolates studied in 2017 (8554/M/17 from patient No.1 and 8567/M/17 from patient No.2) had identical biochemical profiles. A comparison of research results using multiplex PCR from 2017 with a genetic analysis of strains from 2015 enabled us to demonstrate that the strains currently studied have the genes encoding the same toxins (α and β2) as the two strains analyzed in 2015: no.7143 (patient No.3) and no.7149 (patient No.2). A comparative analysis of the strain profiles obtained with pulsed-field gel electrophoresis (PFGE)in 2017 with the results from 2015 has found one identical and genetically unique restriction profile, corresponding to one clone of C. perfringens comprising of two strains: no.8567/M/17 (patient No.2 in 2017) and no.7143 (patient No.3 in 2015). The epidemiological data and detailed analysis of the course of both events suggest that this clone of C. perfringens possibly survived in adverse conditions of the external environment in the operating block of this hospital for many months.

CReasPy-cloning: a method for simultaneous cloning and engineering of megabase-sized genomes in yeast using the CRISPR-Cas9 system

ABSTRACTOver the last decade a new strategy was developed to bypass the difficulties to genetically engineer some microbial species by transferring (or “cloning”) their genome into another organism that is amenable to efficient genetic modifications and therefore acts as a living workbench. As such, the yeastSaccharomyces cerevisiaehas been used to clone and engineer genomes from viruses, bacteria and algae. The cloning step requires the insertion of yeast genetic elements within the genome of interest, in order to drive its replication and maintenance as an artificial chromosome in the host cell. Current methods used to introduce these genetic elements are still unsatisfactory, due either to their random nature (transposon) or the requirement for unique restriction sites at specific positions (TAR cloning). Here we describe the CReasPy-Cloning, a new method that combines both the ability of Cas9 to cleave DNA at a user-specified locus and the yeast’s highly efficient homologous recombination to simultaneously clone and engineer a bacterial chromosome in yeast. Using the 0.816 Mbp genome ofMycoplasma pneumoniaeas a proof of concept, we demonstrate that our method can be used to introduce the yeast genetic element at any location in the bacterial chromosome while simultaneously deleting various genes or group of genes. We also show that CReasPy-cloning can be used to edit up to three independent genomic loci at the same time with an efficiency high enough to warrant the screening of a small (<50) number of clones, allowing for significantly shortened genome engineering cycle times.

Engineering a Minimal Cloning Vector from a pUC18 Plasmid Backbone with an Extended Multiple Cloning Site

Minimal plasmids play an essential role in many intermediate steps in molecular biology. They can for example be used to assemble building blocks in synthetic biology or be used as intermediate cloning plasmids that are ideal for PCR-based mutagenesis methods. A small backbone also opens up for additional unique restriction enzyme cloning sites. Here we describe the generation of pICOz, a 1185 bp fully functional high-copy cloning plasmid with an extended multiple cloning site (MCS). To our knowledge, this is the smallest high-copy cloning vector ever described.

Cloning of sequence of homologous crt-cluster in Streptomyces globisporus 1912-бп

Aim. The aim was to set influence to the additional homologous crt-cluster on carotenogenesis of cells of streptomycete recipient. Methods. For this purpose transformation by the hybrid plasmid pWC 9,6 was conducted. This plasmid contained the fragment of the crt-cluster sequence (9576 bp) of Crt+-mutant S. globisporus Crt4 in a Crt--recipient S. globisporus 1912-бп. To construct this hybrid plasmid, a fragment of PLR-copies of sequence of the crt-cluster of mutant S. globisporus 1912 Crt4 was cloned in the shuttle vector pWHM4 (6.6 kb). Insertion was done into unique restriction sites for endonucleases XbaI and HindIII in a polylinker of this vector. These endonucleases have not restriction sites into the crt-cluster sequence. Results. The plasmid pWC 9,6 (16.2 kb) that contains the crt-cluster sequence (9576 bp) of the Crt+-variant Crt4 of the strain S. globisporus 1912 was constructed. The plasmid successfully functions in the cells of both recipients (E. coli XL1 Blue and S. globisporus 1912-бп). It provides to them resistance to the corresponding antibiotics. The plasmid pWC 9,6 stably keeps its molecular size (16.2 kb). However, indisputable proofs of expression of the crt-clusters in transformants were not got. Conclusions. The plasmid pWC 9,6, that is able to transform and stably function in the cells of both recipient microorganisms (Streptomyces and E. coli) was constructed. Keywords: crt-cluster, shuttle vector, cloning, resistance, PCR.

Engineering a Minimal 1185 Bp Cloning Vector from a Puc18 Plasmid Backbone with an Extended Multiple Cloning Site

Minimal plasmids play an essential role in many intermediate steps in molecular biology. They can for example be used to assemble building blocks in synthetic biology or be used as intermediate cloning plasmids that are ideal for PCR-based mutagenesis methods. A small backbone also opens up for additional unique restriction enzyme cloning sites. Here we describe the generation of a ~1kb fully functional cloning plasmid with an extended multiple cloning site (MCS). To our knowledge, this is the smallest high-copy cloning vector ever described.