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Frontiers in Medicine 2021Bacterial keratitis is a serious and vision-threatening condition in veterinary and human patients, one that often requires culture and susceptibility testing to adjust...
Bacterial keratitis is a serious and vision-threatening condition in veterinary and human patients, one that often requires culture and susceptibility testing to adjust therapy and improve clinical outcomes. The present study challenges the antimicrobial susceptibility testing (AST) paradigm in ophthalmology, enabling more accurate -to- translation by incorporating factors normally present during host-pathogen interactions in clinical patients. Thirty bacteria (10 , 10 , 10 ) were isolated from canine patients with infectious keratitis. For each isolate, commercial plates (Sensititre™ JOEYE2) were used to assess the minimal inhibitory concentration (MIC) of 17 different antibiotics in the absence (0% albumin, control) or presence of canine albumin (0.01-2%). For , the experiment was repeated with actual tear fluid collected from canine eyes with ocular surface inflammation. Kruskal-Wallis, Wilcoxon signed rank test and Spearman's correlation tests were used for statistical analysis. Clinical outcomes were unfavorable in selected canine patients with bacterial keratitis (e.g., globe perforation, graft dehiscence) despite standard AST (i.e., 0% albumin in test medium) confirming that most corneal infections (93%) were susceptible to ≥1 topical antibiotics used at the initial visit. Albumin levels ≥0.05% increased MICs in a dose-dependent, bacteria-specific, and antibiotic-specific manner. No significant differences ( = 1.000) were noted in MICs of any antibiotic whether albumin or tear fluid was added to the Mueller-Hinton broth. Percent protein binding inherent to each antibiotic was associated with clinical interpretations (Spearman's rho = -0.53, = 0.034) but not changes in MICs. Albumin in tears impacted the efficacy of selected ophthalmic antibiotics as only the unbound portion of an antibiotic is microbiologically active. The present findings could improve decision making of clinicians managing bacterial keratitis, reduce development of antimicrobial resistance, influence current guidelines set by the Clinical and Laboratory Standards Institute, and serve as a reference for bacteriological evaluations across medical fields and across species.
PubMed: 34917625
DOI: 10.3389/fmed.2021.663212 -
Japanese Journal of Infectious Diseases May 2022Using bacterial pan-genomes obtained through whole genome sequencing (WGS), coding DNA sequences (CDSs) can be clustered into pan-genome orthologous groups (POGs). We...
Using bacterial pan-genomes obtained through whole genome sequencing (WGS), coding DNA sequences (CDSs) can be clustered into pan-genome orthologous groups (POGs). We aimed to investigate comparative genomic features of Streptococcus canis based on POG analysis and to determine CDSs specific to prevalent sequence type (ST) 9. Twenty WGS datasets from S. canis strains, including invasive and non-invasive specimens, were retrieved from the National Center for Biotechnology Information Assembly database. Based on the WGS data, we performed comparative genome hybridization (CGH), pan- and core-genome prediction, Venn diagram testing with five ST9 strains, and phylogenetic analysis with ST determination. We compared the CDSs of seven ST9 and 13 non-ST9 strains. We observed genomic diversity based on CGH and Venn diagram analyses. The predicted pan- and core-genomes contained 4,772 and 1,403 genes, respectively. We found five clades consisting of different STs (ST1, ST44/2, ST13/14, ST21/15/41, and ST9) based on the phylogenetic tree. There were differences in four pathways (DNA restriction-modification system, DNA-mediated transposition, extracellular region, and response to oxidative stress) regulated by CDSs specific to ST9. Our findings describe genomic diversity in CGH and Venn diagram testing, pan- and core-genomes, five clades of genomes consisting of different STs, and unique CDS features associated with ST9.
Topics: DNA; Genomics; Phylogeny; Streptococcus
PubMed: 34588372
DOI: 10.7883/yoken.JJID.2021.533 -
ACS Synthetic Biology Oct 2021Genome recoding enables incorporating new functions into the DNA of microorganisms. By reassigning codons to noncanonical amino acids, the generation of new-to-nature...
Genome recoding enables incorporating new functions into the DNA of microorganisms. By reassigning codons to noncanonical amino acids, the generation of new-to-nature proteins offers countless opportunities for bioproduction and biocontainment in industrial chassis. A key bottleneck in genome recoding efforts, however, is the low efficiency of recombineering, which hinders large-scale applications at acceptable speed and cost. To relieve this bottleneck, we developed ReScribe, a highly optimized recombineering tool enhanced by CRISPR-Cas9-mediated counterselection built upon the minimal PAM 5'-NNG-3' of the Cas9 (ScCas9). As a proof of concept, we used ReScribe to generate a minimally recoded strain of the industrial chassis by replacing TAG stop codons (functioning as PAMs) of essential metabolic genes with the synonymous TAA. We showed that ReScribe enables nearly 100% engineering efficiency of multiple loci in , opening promising avenues for genome editing and applications thereof in this bacterium and beyond.
Topics: CRISPR-Cas Systems; DNA, Single-Stranded; Escherichia coli; Gene Editing; Genes, Bacterial; Pseudomonas putida; Recombination, Genetic
PubMed: 34547891
DOI: 10.1021/acssynbio.1c00297 -
Cells Aug 2021The CRISPR/Cas9 system has been widely used for gene editing in zebrafish. However, the required NGG protospacer adjacent motif (PAM) of Cas9 (SpCas9) notably restricts...
The CRISPR/Cas9 system has been widely used for gene editing in zebrafish. However, the required NGG protospacer adjacent motif (PAM) of Cas9 (SpCas9) notably restricts the editable range of the zebrafish genome. Recently, Cas9 from (ScCas9), which has a more relaxed 5'-NNG-3' PAM, was reported to have activities in human cells and plants. However, the editing ability of ScCas9 has not been tested in zebrafish. Here we characterized and optimized the activity of ScCas9 in zebrafish. Delivered as a ribonucleoprotein complex, ScCas9 can induce mutations in zebrafish. Using the synthetic modified crRNA:tracrRNA duplex instead of in vitro-transcribed single guide RNA, the low activity at some loci were dramatically improved in zebrafish. As far as we know, our work is the first report on the evaluation of ScCas9 in animals. Our work optimized ScCas9 as a new nuclease for targeting relaxed NNG PAMs for zebrafish genome editing, which will further improve genome editing in zebrafish.
Topics: Animals; CRISPR-Associated Protein 9; CRISPR-Cas Systems; Clustered Regularly Interspaced Short Palindromic Repeats; Gene Editing; Genome; Mutation; Nucleotide Motifs; RNA, Guide, CRISPR-Cas Systems; Streptococcus; Zebrafish; Zebrafish Proteins
PubMed: 34440868
DOI: 10.3390/cells10082099 -
Journal of Integrative Plant Biology Sep 2021Streptococcus canis Cas9 (ScCas9) is an RNA-guided endonuclease with NNG protospacer adjacent motif (PAM) specificity whose genome-editing activity in rice is...
Streptococcus canis Cas9 (ScCas9) is an RNA-guided endonuclease with NNG protospacer adjacent motif (PAM) specificity whose genome-editing activity in rice is locus-dependent. Here we investigated the performance of a ScCas9 variant named Sc at different NNG PAM sites in the rice genome; Sc harbors a T1227K mutation and the substitution of a positively charged loop (residues 367-376). Sc nuclease achieved broader genome editing compared to the original ScCas9, and its nickase improved targeted base editing in transgenic rice plants. Using the high-efficiency adenine base editor rBE73b, we generated many new OsGS1 alleles suitable for screening of rice germplasm for potential herbicide resistance in the future. The CRISPR/Sc system expands the genome-editing toolkit for rice.
Topics: CRISPR-Associated Protein 9; CRISPR-Cas Systems; Gene Editing; Genome, Plant; Oryza; Streptococcus
PubMed: 34427973
DOI: 10.1111/jipb.13166 -
Journal of Integrative Plant Biology Sep 2021The development of clustered regularly interspaced palindromic repeats (CRISPR)-associated protein (Cas) variants with a broader recognition scope is critical for...
The development of clustered regularly interspaced palindromic repeats (CRISPR)-associated protein (Cas) variants with a broader recognition scope is critical for further improvement of CRISPR/Cas systems. The original Cas9 protein from Streptococcus canis (ScCas9) can recognize simple NNG-protospacer adjacent motif (PAM) targets, and therefore possesses a broader range relative to current CRISPR/Cas systems, but its editing efficiency is low in plants. Evolved ScCas9 and ScCas9 variants have been shown to possess higher editing efficiencies in human cells, but their activities in plants are currently unknown. Here, we utilized codon-optimized ScCas9, ScCas9 and ScCas9 and a nickase variant ScCas9n to systematically investigate genome cleavage activity and cytidine base editing efficiency in rice (Oryza sativa L.). This analysis revealed that ScCas9 has higher editing efficiency than ScCas9 and ScCas9 in rice. Furthermore, we fused the evolved cytidine deaminase PmCDA1 with ScCas9n to generate a new evoBE4max-type cytidine base editor, termed PevoCDA1-ScCas9n . This base editor achieved stable and efficient multiplex-site base editing at NNG-PAM sites with wider editing windows (C -C ) and without target sequence context preference. Multiplex-site base editing of the rice genes OsWx (three targets) and OsEui1 (two targets) achieved simultaneous editing and produced new rice germplasm. Taken together, these results demonstrate that ScCas9 represents a crucial new tool for improving plant editing.
Topics: CRISPR-Associated Protein 9; CRISPR-Cas Systems; Gene Editing; Oryza; Streptococcus
PubMed: 34411422
DOI: 10.1111/jipb.13164 -
International Journal of Molecular... Jul 2021Methylprednisolone is a glucocorticoid and can negatively influence immune defense mechanisms. During bacterial infections in the dog, neutrophils infiltrate infected...
Methylprednisolone is a glucocorticoid and can negatively influence immune defense mechanisms. During bacterial infections in the dog, neutrophils infiltrate infected tissue and mediate antimicrobial effects with different mechanisms such as phagocytosis and neutrophil extracellular trap (NET) formation. Here, we investigated the influence of methylprednisolone on canine NET formation and neutrophil killing efficiency of Gram positive and Gram negative bacteria. Therefore, canine blood derived neutrophils were treated with different concentrations of methylprednisolone over time. The survival factor of , or was determined in presence of stimulated neutrophils. Additionally, free DNA and nucleosomes as NET marker were analyzed in supernatants and neutrophils were assessed for NET formation by immunofluorescence microscopy. Methylprednisolone concentrations of 62.5 and 625 µg/mL enhanced the neutrophil killing of Gram positive bacteria, whereas no significant influence was detected for the Gram negative . Interestingly, higher amounts of free DNA were detected under methylprednisolone stimulation in a concentration dependency and in the presence of and . The nucleosome release by neutrophils is induced by bacterial infection and differs depending on the concentration of methylprednisolone. Furthermore, immunofluorescence microscopy analysis identified methylprednisolone at a concentration of 62.5 µg/mL as a NET inducer. In summary, methylprednisolone enhances NET-formation and time-dependent and concentration-dependent the bactericidal effect of canine neutrophils on Gram positive bacteria.
Topics: Animals; Anti-Bacterial Agents; Dogs; Extracellular Traps; Female; Gram-Negative Bacteria; Gram-Positive Bacteria; Male; Methylprednisolone; Neutrophils; Nucleosomes; Phagocytosis
PubMed: 34299355
DOI: 10.3390/ijms22147734 -
Japanese Journal of Infectious Diseases Jan 2022This study assessed the biofilm production ability (BPA) and other microbiological features of Streptococcus canis strains. Forty strains of companion-animal origin,...
This study assessed the biofilm production ability (BPA) and other microbiological features of Streptococcus canis strains. Forty strains of companion-animal origin, including the host information, from 2015 and 2017 were randomly selected, and three strains of blood-origin from two humans and one dog were included. We measured BPA using crystal violet staining, along with S. canis M-like protein (SCM) allele typing, sequence type (ST) determination, antimicrobial resistance (AMR) phenotyping/genotyping, and virulence-associated gene profiling (gbp, ap1, fp1, and brp). BPA measurements revealed 35 strains with BPA and 48 strains without BPA. There was an association between the producer and the isolation year (2017). Moreover, we observed an association between the non-producer and SCM allele 1 and ST9, and between the producer and SCM allele 10 and ST21. Furthermore, we observed a correlation between the producer and the presence of AMR genotypes. Specifically, there was an association between the producer and ap1 detection, and between non-producer and gbp detection. Our results suggest a correlation between biofilm producers and other microbiological features (i.e. isolation year, SCM allele type 10, ST21, presence of AMR genotypes, and ap1 detection).
Topics: Animals; Biofilms; Dogs; Genotype; Streptococcal Infections; Streptococcus
PubMed: 34193659
DOI: 10.7883/yoken.JJID.2020.1086 -
Antibiotics (Basel, Switzerland) May 2021Antimicrobial stewardship programs (ASPs) have been suggested to reduce antimicrobial resistance phenomena in veterinary medicine, as antibiotics are commonly used...
Antimicrobial stewardship programs (ASPs) have been suggested to reduce antimicrobial resistance phenomena in veterinary medicine, as antibiotics are commonly used without microbiological confirmation. The aim of the present study is to design a specific working flow for a tailored antimicrobial treatment in the case of canine and feline urinary tract infections (UTIs). Urine samples were collected by cystocentesis from 16 dogs and 12 cats presenting acute signs of UTI. The therapy was decided according to the minimal inhibitory concentration, and it was possible to monitor 14 dogs and 11 cats. Rescue therapy (amoxicillin and clavulanic acid) was included in emergency cases. , , and were isolated in dogs, and , , and were isolated in cats. No multidrug-resistant strains were detected, but all were methicillin resistant. Only one cat received rescue therapy, and only one dog was recruited. Dogs were treated with tetracycline (1/14), fluoroquinolones (6/14), beta-lactams (6/14), and gentamicin (1/14), while cats received fluoroquinolones (3/11), nitrofurans (1/11), clindamycin (1/11), and beta-lactams (6/11). The success rate was very high. Our findings are interesting because this is the first ASP in Italy, and it may be used as a model to develop ASPs for other pathologies.
PubMed: 34064943
DOI: 10.3390/antibiotics10050562 -
Arthroplasty Today Apr 2021A 61-year-old man underwent elective primary total hip arthroplasty at an academic center and presented to the emergency department 2 weeks later with a periprosthetic...
A 61-year-old man underwent elective primary total hip arthroplasty at an academic center and presented to the emergency department 2 weeks later with a periprosthetic infection. Intraoperative cultures were positive for He was successfully treated with one-stage revision and 6 weeks of intravenous cefazolin. It was later determined that the patient has a pet dog who frequently licks his legs. We hypothesize that patients with pets are more likely to carry this pathogen as part of their skin microbiome, and further research is required to establish whether poses an infectious risk beyond that of normal group B Streptococcus skin flora and if preoperative decolonization strategies are warranted.
PubMed: 33718556
DOI: 10.1016/j.artd.2021.01.010