Gene Cassette Research Articles

Genetic and phenotypic analyses of mcr-harboring extended-spectrum β-lactamase-producing Escherichia coli isolates from companion dogs and cats in Japan

The emergence of mcr plasmid-mediated colistin-resistant extended-spectrum β-lactamase (ESBL)-producing Enterobacterales among companion dogs and cats poses a risk of the animals acting as reservoirs for cross-species transmission. However, current knowledge of mcr-harboring ESBL-producing Enterobacterales in companion dogs and cats is still limited; thus, the genetic and phenotypic characteristics of the bacterial isolates and plasmids, in companion dogs and cats, remain to be elucidated. Here, we identified mcr gene-harboring ESBL-producing Escherichia coli isolates during whole-genome sequencing of ESBL-producing E. coli isolates from a dog and a cat in Osaka, Japan. Colistin-resistant MY732 isolate from a dog carried two plasmids: mcr-1.1-harboring IncI2 plasmid and blaCTX-M-14-harboring IncFIB plasmid. Conjugation assays revealed that both plasmids can be co-transferred even though the IncFIB plasmid lacked a conjugal transfer gene cassette. The other isolate MY504 from a cat harbored two bla genes and mcr-9 on the identical IncHI2 plasmid. This isolate was not resistant to colistin, which is likely to be due to deletion of the regulatory two-component QseBC system associated with the mcr-9 expression. To the best of our knowledge, this is the first report of a colistin-resistant ESBL-producing E. coli isolate harboring mcr-1 from a companion dog in Japan. Given that the mcr gene-harboring IncI2 and IncHI2 plasmids in this study shared high homology with plasmids from human or animal-derived Enterobacterales, companion dogs and cats may act as important reservoirs for cross-species transmission of the mcr gene in the community, in Japan.

Effects of antibiotics on the in vitro expression of tetracycline-off constructs and the performance of Drosophila suzukii female-killing strains.

Genetic control strategies such as the Release of Insects Carrying a Dominant Lethal (RIDL) gene and Transgenic Embryonic Sexing System (TESS) have been demonstrated in the laboratory and/or deployed in the field. These strategies are based on tetracycline-off (Tet-off) systems which are regulated by antibiotics such as Tet and doxycycline (Dox). Here, we generated several Tet-off constructs carrying a reporter gene cassette mediated by a 2A peptide. Different concentrations (0.1, 10, 100, 500, and 1,000μg/mL) and types (Tet or Dox) of antibiotics were used to evaluate their effects on the expression of the Tet-off constructs in the Drosophila S2 cells. One or both of the two concentrations, 100 and 250μg/mL, of Tet or Dox were used to check the influence on the performances of a Drosophila suzukii wild-type strain and female-killing (FK) strains employing TESS. Specifically, the Tet-off construct for these FK strains contains a Drosophila suzukii nullo promoter to regulate the tetracycline transactivator gene and a sex-specifically spliced pro-apoptotic gene hid Ala4 to eliminate females. The results suggested that the in vitro expression of the Tet-off constructs was controlled by antibiotics in a dose-dependent manner. ELISA experiments were carried out identifying Tet at 34.8ng/g in adult females that fed on food supplemented with Tet at 100μg/mL. However, such method did not detect Tet in the eggs produced by antibiotic-treated flies. Additionally, feeding Tet to the parents showed negative impact on the fly development but not the survival in the next generation. Importantly, we demonstrated that under certain antibiotic treatments females could survive in the FK strains with different transgene activities. For the strain V229_M4f1 which showed moderate transgene activity, feeding Dox to fathers or mothers suppressed the female lethality in the next generation and feeding Tet or Dox to mothers generated long-lived female survivors. For the strain V229_M8f2 which showed weak transgene activity, feeding Tet to mothers delayed the female lethality for one generation. Therefore, for genetic control strategies employing the Tet-off system, the parental and transgenerational effects of antibiotics on the engineered lethality and insect fitness must be carefully evaluated for a safe and efficient control program.

The linkage between prevalence of integron I and reduced susceptibility to biocides in MDR Klebsiella pneumoniae isolated from neonates.

Klebsiella pneumoniae causes challenging nosocomial fatal infections including neonatal sepsis. Our study aims at clarifying the contribution of integrons in the observed reduced susceptibility of multidrug-resistant (MDR) K. pneumoniae isolated from septicemic neonates to the clinically used antimicrobial agents and biocides. Eighty-six K. pneumoniae isolates were collected from Mansoura University Children's Hospital from septicemic neonates. Isolates were subjected to antibiotic and biocide susceptibility using disk diffusion and the agar dilution method, respectively. The distribution of different classes of integrons was screened in the isolates by PCR. Detected inegron I was sequenced in selected isolates. Fifty-seven isolates (66.27%) were MDR. In the MDR isolates, class I integron was detected in 23 (40.3%), integron III was detected in 20 (35%), whereas integron II could not be detected. Sequencing results of integron I from MDR K. pneumoniae isolates revealed that only aminoglycoside and folate synthesis inhibitors gene cassettes were detected, while the rest of the resistance genes were not associated with integron I. The presence of integron I in MDR K. pneumoniae tested isolates may contribute only to some biocide resistance, however, it does not seem to be the only contributor in multiple drug resistance.

Global profiling of antibiotic resistomes in maize rhizospheres

The spreading of antimicrobial resistance (AMR) in crops and food products represents a global concern. In this study, we conducted a survey of resistomes in maize rhizosphere from Michigan, California, the Netherlands, and South Africa, and investigated potential associations with host bacteria and soil management practices in the crop field. For comparison, relative abundance of antibiotic resistance genes (ARGs) is normalized to the size of individual metagenomes. Michigan maize rhizosphere metagenomes showed the highest abundance and diversity of ARGs, with the detection of blaTEM-116, blaACT-4/-6, and FosA2, exhibiting high similarity (≥ 99.0%) to those in animal and human pathogens. This was probably related to the decade-long application of manure/composted manure from antibiotic-treated animals. Moreover, RbpA, vanRO, mtrA, and dfrB were prevalently found across most studied regions, implying their intrinsic origins. Further analysis revealed that RbpA, vanRO, and mtrA are mainly harbored by native Actinobacteria with low mobility since mobile genetic elements were rarely found in their flanking regions. Notably, a group of dfrB genes are adjacent to the recombination binding sites (attC), which together constitute mobile gene cassettes, promoting the transmission from soil bacteria to human pathogens. These results suggest that maize rhizosphere resistomes can be distinctive and affected by many factors, particularly those relevant to agricultural practices.

A novel technique for large-fragment knock-in animal production without ex vivo handling of zygotes

CRISPR/Cas-based genome editing has dramatically improved genetic modification technology. In situ electroporation called genome editing via oviductal nucleic acid delivery (GONAD), which eliminates the need for ex vivo embryo handling, is technically the simplest method for gene transfer and can be performed in laboratories without developmental engineering expertise including micromanipulation techniques. However, the use of this method remains challenging in the case of large-fragment knock-in, such as gene expression cassettes. Adeno-associated viruses (AAV) act as donor DNA for homologous recombination in infected cells, including rodent embryos. In this study, we demonstrated simultaneous electroporation of AAV donors and CRISPR/Cas9 components into embryos to create knock-in animals, and successfully generated knock-in rats carrying a gene cassette with a length of 3.0 kb using a small number of animals and in situ electroporation. These findings indicate that this technique is an efficient high-throughput strategy for producing genetically modified rodents and may be applicable to other animal species.

High Occurrence of Multiple-Drug Resistance Mediated by Integron in Aeromonas Isolated from Fish-Livestock Integrated Farms

Fish-livestock integrated farming is a traditional practice in South China and Southeast Asia. The administration of antimicrobial agents in livestock may potentially facilitate the spread of antimicrobial resistance to aquaculture through the drainage of livestock manure. Aeromonas spp. are fish pathogens that are the predominant bacteria in water bodies. The aim of this study was to characterize multiple-drug-resistantAeromonas spp. isolated from integrated and nonintegrated fish-livestock farms and to investigate the occurrence of class 1 integrons. Of the 481 Aeromonas strains examined in this study, isolates from pig manure, fish, and environmental samples from pig-fish or goose-fish integrated farms were significantly more resistant to various antimicrobial agents than those from nonintegrated farms P < 0.05 . High rates of resistance to nalidixic acid and sulfamonomethoxine were observed in the isolates from the integrated farm. Fifty Aeromonas isolates (10.4%) from integrated farms contained class 1 integrons. In addition, 96.0% (48/50) of the integron-positive strains displayed multiple-drug resistance. Ten types of gene cassette arrays were determined by sequencing, including dfrA17, dfrA12-orfF-aadA2, catB8, dfrB4-catB3-aadA1, aac6(6′)-Ib-cr-arr-3, aac-II-blaOXA-21-catB3, aar2-aacA4-drfA1-orfC, aac(6′)-Ib-cr, dfrA15, and dfrB4-catB3-blaOXA-10-aadA1. Notably, among the 50 integron-positive isolates, twenty isolates showed integrons located in plasmids, which may facilitate the transmission of resistance in integrated farms. Our investigation confirmed the high prevalence of multiple antimicrobial resistances mediated by class 1 integrons in Aeromonas isolates from integrated farms. Therefore, it is necessary to establish a risk assessment method for antimicrobial resistance in aquaculture.

Prevalence of Class 1 Integron and In Vitro Effect of Antibiotic Combinations of Multidrug-Resistant Enterococcus Species Recovered from the Aquatic Environment in the Eastern Cape Province, South Africa.

Enterococci are regarded as a better indication of faecal pollution in freshwater and marine waters. Their levels in seawater are positively connected with swimming-related gastrointestinal disorders. This study used an Enterococcus-specific polymerase chain reaction (PCR) to characterize the isolates. Classes 1 and 2 integrons were examined for environmental Enterococcus isolates using a standard biological procedure. All strains were assessed against a panel of 12 antibiotics from various classes using disc diffusion methods. The microdilution method was used to work out the minimum inhibitory concentration (MIC) according to the CLSI guiding principles. The combination therapy of the resistant drugs was evaluated using a checkerboard assay and a time-dependent test for assessing their bactericidal or bacteriostatic activity. The gene diversity of the tested organisms was analyzed with the aid of Enterobacterial Repetitive Intergenic Consensus (ERIC) PCR. In total, 57 Enterococcus spp. environmental samples were recovered, in which Enterococcus faecalis (33.33%) and Enterococcus faecium (59.65%) were the dominant species. Resistance to linezolid, ciprofloxacin, erythromycin, gentamicin, vancomycin, rifampicin, and tetracycline was prevalent. Fifty (50) strains tested positive for class 1 integron, more frequent in Enterococcus faecium and Enterococcus faecalis isolates, with no gene cassette array discovered. A combination of gentamicin (MIC 4 µg/mL) with vancomycin (MIC 256 µg/mL) antibiotics against Enterococcus faecalis showed antibacterial activity. In contrast, the combination of ciprofloxacin (1 µg/mL) with Ampicillin (16 µg/mL) antibiotics against Enterococcus faecalis showed a bacteriostatic effect. The ERIC-PCR analysis pointed out that most of the assessed isolates have close genetic similarities.

Bioinformatics design of recombinant chimeric protein containing SipD and LptD immunogens and evaluation of its immunogenicity against Salmonella Typhimurium

The growing emergence of resistant bacteria is the current global concern for the humans and animals. Vaccination could be the desirable method to preventing such infectious diseases. Safe and effective vaccines are urgently needed to manage and prevent Salmonella contamination. Subunit vaccines are safe approaches for the protection against Salmonella spp. The bioinformatics methods were performed to determine the gene structure. Gene cassette (rLPSI) was ordered in pET28a (+), and cloned into E.coli BL21 (DE3), and the recombinant protein was expressed using IPTG (1mM). Mice were immunized by subcutaneous administration of recombinant protein. Then, the mice were challenged by oral administration of 100LD50 of live S. Typhimurium. The Codon adaptation index of the chimeric gene was multiplied by 0.92. Validation results showed that >90% of residues lie in the desired or extra allowed area of the Ramachandran plot. The recombinant protein (65.9kDa) was expressed in E.coli. Antibody titers in vaccinated mice were significantly different from those in the control groups. Recombinant protein immunization of the mice provided 90% and 70% protection against 10LD50 and 100LD50 of S. Typhimurium, respectively. In general, the results showed the high efficiency of rLPSI chimeric protein as a protective antigen against S. Typhimurium infection. The rLPSI chimeric protein could be an effective recombinant vaccine candidate against S. Typhimurium infection, but more research is needed.

The fate of sulfonamide resistance genes and anthropogenic pollution marker intI1 after discharge of wastewater into a pristine river stream.

Currently there are sparse regulations regarding the discharge of antibiotics from wastewater treatment plants (WWTP) into river systems, making surface waters a latent reservoir for antibiotics and antibiotic resistance genes (ARGs). To better understand factors that influence the fate of ARGs in the environment and to foster surveillance of antibiotic resistance spreading in such habitats, several indicator genes have been proposed, including the integrase gene intI1 and the sulfonamide resistance genes sul1 and sul2. Here we used quantitative PCR and long-read nanopore sequencing to monitor the abundance of these indicator genes and ARGs present as class 1 integron gene cassettes in a river system from pristine source to WWTP-impacted water. ARG abundance was compared with the dynamics of the microbial communities determined via 16S rRNA gene amplicon sequencing, conventional water parameters and the concentration of sulfamethoxazole (SMX), sulfamethazine (SMZ) and sulfadiazine (SDZ). Our results show that WWTP effluent was the principal source of all three sulfonamides with highest concentrations for SMX (median 8.6 ng/l), and of the indicator genes sul1, sul2 and intI1 with median relative abundance to 16S rRNA gene of 0.55, 0.77 and 0.65%, respectively. Downstream from the WWTP, water quality improved constantly, including lower sulfonamide concentrations, decreasing abundances of sul1 and sul2 and lower numbers and diversity of ARGs in the class 1 integron. The riverine microbial community partially recovered after receiving WWTP effluent, which was consolidated by a microbiome recovery model. Surprisingly, the relative abundance of intI1 increased 3-fold over 13 km of the river stretch, suggesting an internal gene multiplication. We found no evidence that low amounts of sulfonamides in the aquatic environment stimulate the maintenance or even spread of corresponding ARGs. Nevertheless, class 1 integrons carrying various ARGs were still present 13 km downstream from the WWTP. Therefore, limiting the release of ARG-harboring microorganisms may be more crucial for restricting the environmental spread of antimicrobial resistance than attenuating ng/L concentrations of antibiotics.

Antibiotic resistance profile and molecular characterization of extraintestinal pathogenic Escherichia coli (ExPEC) from human clinical samples in gaza strip, palestine.

The rates of antibiotic resistance in extraintestinal pathogenic Escherichia coli (ExPEC) have increased significantly in recent years. This study aims at studying antibiotic resistance, virulence factors (VFs), and the phylogenetic background of ExPEC isolated from Palestinian patients. A total of 42 ExPEC isolates were collected from patients with extraintestinal infections in three Palestinian hospitals. Antimicrobial susceptibility was studied by the disk diffusion method. Resistance/virulence-gene profiles, phylogenetic groups, and integron profiles of these isolates were studied by PCR. The susceptibility to carbapenems was detected in 90.5% of the isolates. Half of the isolates were resistant to ampicillin and sulfamethoxazole/trimethoprim, and 33.3% of the isolates were multidrug-resistant. BlaTEM-1 was detected in seven isolates and blaOXA-1 was identified in one isolate. Sul, qnrA, and aac(6')-Ib-cr genes were found in 12, 3, and 2 isolates, respectively. Class 1 integron has been identified in 10 isolates with the gene cassette arrangement dhfr17+aadA5. The isolates were distributed between phylogroups B2 and D. The presence of VFs, antibiotic resistance genes, and class 1 integron associated with phylogenetic groups (B2 and D) among multidrug-resistant (MDR)-ExPEC recovered from urinary tract infections (UTIs) patients will complicate infection management and increase therapy failure. Routine screening of antibiotic resistance is important for appropriate and efficient empirical treatment.

Identification of integrons and gene cassette-associated recombination sites in bacteriophage genomes.

Bacteriophages are versatile mobile genetic elements that play key roles in driving the evolution of their bacterial hosts through horizontal gene transfer. Phages co-evolve with their bacterial hosts and have plastic genomes with extensive mosaicism. In this study, we present bioinformatic and experimental evidence that temperate and virulent (lytic) phages carry integrons, including integron-integrase genes, attC/attI recombination sites and gene cassettes. Integrons are normally found in Bacteria, where they capture, express and re-arrange mobile gene cassettes via integron-integrase activity. We demonstrate experimentally that a panel of attC sites carried in virulent phage can be recognized by the bacterial class 1 integron-integrase (IntI1) and then integrated into the paradigmatic attI1 recombination site using an attC x attI recombination assay. With an increasing number of phage genomes projected to become available, more phage-associated integrons and their components will likely be identified in the future. The discovery of integron components in bacteriophages establishes a new route for lateral transfer of these elements and their cargo genes between bacterial host cells.

Comparative genetic characterization of CMY-2-type beta-lactamase producing pathogenic Escherichia coli isolated from humans and pigs suffering from diarrhea in Korea

BackgroundPathogenic Escherichia coli are an important cause of bacterial infections in both humans and pigs and many of antimicrobials are used for the treatment of E. coli infection. The objective of this study was to investigate the characteristics and relationship between humans and pigs regarding third-generation cephalosporin resistance and CMY-2-producing E. coli in Korea.ResultsAll 103 third-generation cephalosporin-resistant E. coli isolates showed multidrug resistance. Also, except for β-lactam/β-lactamase inhibitor combinations, all antimicrobials resistant rates were higher in pigs than in humans. A total of 36 isolates (humans: five isolates; pigs: 31 isolates) were positive for the CMY-2-encoding genes and thirty-two (88.9%) isolates detected class 1 integrons with 10 different gene cassette arrangements, and only 1 isolate detected a class 2 integron. The most common virulence genes in pigs were LT (71.0%), F18 (51.6%), and STb (51.6%), while stx2 (80.0%) was the most frequently detected gene in humans. Stx2 gene was also detected in pigs (6.5%). Interestingly, 36 CMY-2-producing E. coli isolates showed a high diversity of sequence types (ST), and ST88 was present in E. coli from both pigs (11 isolates) and humans (one isolate).ConclusionOur findings suggest that a critical need for comprehensive surveillance of third-generation cephalosporin resistance is necessary to preserve the usefulness of third-generation cephalosporins in both humans and pigs.

Molecular insights into novel environmental strains of Klebsiella quasipneumoniae harboring different antimicrobial-resistance genes.

The emergence of bacterial pathogens in environmental hosts represents a major risk to public health. This study aimed at characterizing seven novel environmental strains of K. quasipneumoniae using a genomic approach which was misidentified by phenotypic methods in a previous batch of 27 species thought to be K. pneumoniae. Whole-genome sequencing was performed using the Illumina platform, and the generated raw reads were de novo assembled. Comparative genomic, resistome, virulome, mobilome, and phylogeny were then investigated using dierent bioinformatics tools. Six strains were identified as K. quasipneumoniae subsp similipneumoniae and one as K. quasipneumoniae subsp. quasipneumoniae. All isolates were resistant to ampicillin, cephalexin, and amoxicillin-clavulanic acid and harbored the fosA, bla OKP types, oqxB, and oqxA genes. One isolate additionally harbored a gene cassettes consisting of bla SHV-1, bla OXA-1, aac(6')-Ib-cr, catB genes. The aminoglycoside-modifying enzyme gene aph(3")-Ia was bracketed by two insertion elements. Plasmid analyses showed that IncFIBK was the most prevalent plasmid, circulating in six isolates, while one isolate exhibited seven different plasmids. The isolates have virulence genes responsible for capsule formation, lipopolysaccharide, iron uptake aerobactin (iutA), salmochelins (iroE, iroN), enterobactin siderophore, adherence, and biofilm formation (mrkA, mrkB, mrkC, mrkD, mrkF, and mrkH). Our study highlights the ecology and transmission of K. quasipneumoniae (which have the ability to disseminate to other environmental sources including animals) outside the clinical setting and the contribution of water, vegetables, and table surfaces as potential reservoirs of farm-to-fork transmission of disease via local markets in Khartoum, Sudan.

In planta transformation of Polianthes tuberosa for concomitant knockdown of flp-1, flp-12 and flp-18 genes induced root-knot nematode resistance

Polianthes tuberosa is a lucrative crop owing to its huge demand in floriculture, perfume and pharmaceutical industry. Root-knot nematodes, especially Meloidogyne incognita poses a major impediment on tuberose yield. Currently nematode management relies on the use of hazardous nematicides and other inefficacious means viz. crop rotation and soil solarization. The ongoing search for an ecologically sustainable and target specific approach, led to the progression of genetic modification based techniques to target genes critical for nematode survival. FMRFamide like peptide (FLPs) genes have drawn interest as prospective targets because of their diverse physiological roles in nematodes. Here, the effect of concomitant silencing of three vital nematode FLP genes (Mi-flp-1, Mi-flp-12 and Mi-flp-18) using host induced RNAi in tuberose was assessed against M. incognita. Firstly, the fusion (flp-1, flp-12 and flp-18) gene cassette was functionally validated using host-delivered RNAi in tobacco model system. Transgenic tobacco expressing dsRNA of fusion gene significantly reduced the nematode multiplication factor (MF) by 82.6%. Since, genetic transformation of tuberose has been neglected so far, and transgenic development is an inevitable step for host-generated RNAi, an Agrobacterium mediated transformation protocol was optimized in tuberose. Tuberose tubers were pricked at the apical meristem of the axis and at the tiny white meristematic buds emerging from them, followed by infection with Agrobacterium hosting pCAMBIA 3301 vector. Maximum transformation efficiency was achieved with 200 μM acetosyringone as observed from the GUS staining. For HIGS, fusion transgenic events were developed in tuberose and molecular analysis confirmed the presence and integration of fusion transgene using PCR, qRT-PCR and Southern blot analysis. Bioefficacy analysis of transgenic plants against M. incognita resulted in 73.75% reduction in nematode fecundity, thereby demonstrating the effectiveness of the three flp genes for nematode management in tuberose. This study established first ever report of in planta Agrobacterium-mediated transformation of tuberose for host delivered RNAi for M. incognita resistance.

Class 1 and 2 Integrons in Escherichia coli Strains Isolated from Diarrhea and Bacteremia in Children Less Than 2 Years of Age from Peru.

Class 1 and Class 2 integrons are mobilizable elements able to carry a variety of antibiotic resistance determinants. In the present study, Class 1 and 2 integrons present in 355 pathogenic Escherichia coli (285 diarrheagenic, of these 129 were enteropathogenic, 90 enteroaggregative, 66 enterotoxigenic, and 70 bacteremic) isolated from healthy and ill children under age 5 from periurban areas of Lima, Peru, were characterized. The presence of integrase 1 and 2 was established by polymerase chain reaction (PCR), and variable regions were grouped by PCR-restriction fragment length polymorphism and subsequent sequencing. Antimicrobial resistance was established by disk diffusion. Ninety-seven isolates (27.3%) presented integrase 1, and 16 (4.5%) presented integrase 2 (P < 0.0001); in addition, seven (2.0%) isolates, six diarrheagenic and one bacteremic, presented both integrase genes. The presence of integrase 1 was more frequent among bacteremic isolates (P = 0.0004). Variable regions were amplified in 76/120 (63.3%) isolates with up to 14 gene arrangements. The most prevalent gene cassettes were those encoding dihydrofolate reductases as well as aminoglycoside modifying enzymes. Of note, Class 1 integrons tended to be associated with the presence of extended-spectrum β-lactamases (ESBLs). A variety of Class 1 and 2 integrons were detected in diarrheagenic and bacteremic E. coli, demonstrating the heterogeneity of variable regions circulating in the area. The association of integrons with ESBLs is worrisome and has an impact on the development of multidrug resistance.

Resolving colistin resistance and heteroresistance in Enterobacter species

Species within the Enterobacter cloacae complex (ECC) include globally important nosocomial pathogens. A three-year study of ECC in Germany identified Enterobacter xiangfangensis as the most common species (65.5%) detected, a result replicated by examining a global pool of 3246 isolates. Antibiotic resistance profiling revealed widespread resistance and heteroresistance to the antibiotic colistin and detected the mobile colistin resistance (mcr)−9 gene in 19.2% of all isolates. We show that resistance and heteroresistance properties depend on the chromosomal arnBCADTEF gene cassette whose products catalyze transfer of L-Ara4N to lipid A. Using comparative genomics, mutational analysis, and quantitative lipid A profiling we demonstrate that intrinsic lipid A modification levels are genospecies-dependent and governed by allelic variations in phoPQ and mgrB, that encode a two-component sensor-activator system and specific inhibitor peptide. By generating phoPQ chimeras and combining them with mgrB alleles, we show that interactions at the pH-sensing interface of the sensory histidine kinase phoQ dictate arnBCADTEF expression levels. To minimize therapeutic failures, we developed an assay that accurately detects colistin resistance levels for any ECC isolate.

Oral booster vaccine antigen-Expression of full-length native SARS-CoV-2 spike protein in lettuce chloroplasts.

Oral booster vaccine antigen-Expression of full-length native SARS-CoV-2 spike protein in lettuce chloroplasts.

First Report of IMI-2-Producing Enterobacter bugandensis and CTX-M-55-Producing Escherichia coli isolated from Healthy Volunteers in Tunisia.

The aim of this study was to characterize the prevalence of fecal carriage of extended-spectrum beta-lactamases and carbapenemase-producing Gram-negative bacteria among healthy humans in Tunisia. Fifty-one rectal swabs of healthy volunteers were plated on MacConkey agar plates supplemented with cefotaxime or imipenem. The occurrences of resistance genes, integrons, and phylogroup typing were investigated using PCR and sequencing. The genetic relatedness of isolates was determined by pulsed-field-gel-electrophoresis (PFGE) and multilocus-sequence-typing (MLST). Whole-genome-sequencing (WGS) was performed for the carbapenem-resistant isolate. Sixteen ESBL-producing Escherichia coli isolates and one carbapenem-resistant Enterobacter bugandensis were detected out of the fifty-one fecal samples. The ESBL-producing E. coli strains contained genes encoding CTX-M-15 (n = 9), CTX-M-1 (n = 3), CTX-M-27 (n = 3), and CTX-M-55 (n = 1). Three CTX-M-1-producers were of lineages ST131, ST7366, and ST1158; two CTX-M-15-producers belonged to lineage ST925 and ST5100; one CTX-M-27-producer belonged to ST2887, and one CTX-M-15-producer belonged to ST744. Six isolates contained class 1 integrons with the following four gene cassette arrangements: dfrA5 (two isolates), dfrA12-orf-aadA2 (two isolates), dfrA17-aadA5 (one isolate), and aadA1 (one isolate). E. bugandensis belonged to ST1095, produced IMI-2 carbapenemase, and contained qnrE1 and fosA genes. A genome-sequence analysis of the E. bugandensis strain revealed new mutations in the blaACT and qnr genes. Our results reveal an alarming rate of ESBL-E. coli in healthy humans in Tunisia and the first description of IMI-2 in E. bugandensis.

Genome-wide identification and expression analysis of the ZF-HD gene family in pea (Pisum sativum L.).

Pea is a conventional grain-feed-grass crop in Tibet and the only high-protein legume in the region; therefore, it plays an important role in Tibetan food and grass security. Zinc finger-homeodomain (ZF-HD) belongs to a family of homozygous heterotypic cassette genes, which play an important role in plant growth, development, and response to adversity stress. Using a bioinformatics approach, 18 PsZF-HD family members were identified. These genes were distributed across seven chromosomes and two scaffold fragments, and evolutionary analysis classified them into two subgroups, MIF and ZHD. The MIF subgroup was subdivided into three subclasses (PsMIFⅠ-III), and the ZHD subgroup was subdivided into five subclasses (ZHDⅠ-V). The PsZF-HD members were named PsMIF1-PsMIF4 and PsZHD1-PsZHD14. Twelve conserved motifs and four conserved domains were identified from PsZF-HD family, of which MIF subgroup only contained one domain, while ZHD subgroup contained two types of domains. In addition, there were significant differences in the three-dimensional structures of the protein members of the two subgroups. Most PsZF-HD genes had no introns (13/18), and only five genes had one intron. Forty-five cis-acting elements were predicted and screened, involving four categories: light response, stress, hormone, and growth and development. Transcriptome analysis of different tissues during pea growth and development showed that PsZHD11, 8, 13, 14 and MIF4 were not expressed or were individually expressed in low amounts in the tissues, while the other 13 PsZF-HDs genes were differentially expressed and showed tissue preference, as seen in aboveground reproductive organs, where PsZHD6, 2, 10 and MIF1 (except immature seeds) were highly expressed. In the aerial vegetative organs, PsZHD6, 1, and 10 were significantly overexpressed, while in the underground root system, PsMIF3 was specifically overexpressed. The leaf transcriptome under a low-nitrogen environment showed that the expression levels of 17 PsZF-HDs members were upregulated in shoot organs. The leaf transcriptome analysis under a low-temperature environment showed stress-induced upregulation of PsZHD10 and one genes and down-regulation of PsZHD6 gene. These results laid the foundation for deeper exploration of the functions of the PsZF-HD genes and also improved the reference for molecular breeding for stress resistance in peas.

Efficient CRISPR/Cas9-Assisted Knockin of Large DNA Donors by Pronuclear Microinjection During S-Phase in Mouse Zygotes.

In the CRISPR/Cas9-mediated gene cassette knockin (KI) strategy, a gene cassette is integrated into a target locus through a proper DNA repair pathway after the Cas9-induced double-strand DNA breaks; the activation of the DNA repair pathway is known to be correlated with the cell cycle. Recently, we have reported a new KI approach named SPRINT (S-phase pronuclear injection for targeting)-CRISPR, focusing on the correlation between the cell cycle and the KI efficiency in the mouse zygote microinjection. Our results suggest that the CRISPR-mediated KI with a homologous recombination-based donor vector during S-phase enhances the KI efficiency. For SPRINT-CRISPR, the uniformity of the zygotes in the cell cycle is achieved by in vitro fertilization, and the zygotes are cryopreserved until use. These reproductive techniques are necessary for efficient KI. Furthermore, Piezo-assisted microinjection has been successful in improving the survival rate of the injected embryos. In this chapter, we describe the protocols that focus on the zygote preparation and Piezo-assisted microinjection of the SPRINT-CRISPR method.