Removal of dye pollution by an oxidase derived from mutagenesis of the Deuteromycete Myrothecium with high potential in industrial applications.

It is estimated that over 700,000 tons of synthetic dyes are produced annually, 15% of which are emitted as effluents. These highly stable dyes enter the world water ecosystems and stay in the environment, and eventually cause adverse impacts to the environment. Current wastewater treatment methods, such as filtration, coagulation, and chemical oxidation, have sideeffects, including toxic residue formation, membrane fouling, bioaccumulation, and secondary pollutant formation. Given the issues mentioned, it is necessary to study how to improve the degradation of synthetic dye with a cost-effective and ecofriendly approach. Natural oxidation provides a greener option. Recently, Deuteromycetes fungus Myrothecium verrucaria G-1 (M. verrucaria G-1) has shown great potential in producing high level of dye oxidase. This study aims to generate a dye oxidase hyperproducer, 3H6 from M. verrucaria G-1 by using atmospheric and room temperature plasma (ARTP) coupled with ultraviolet (UV) irradiation. This method increases oxidase production by nearly 106.15%. After a simple precipitation and dialysis, this mutant oxidase increases by 1.97-fold in a specific activity with dye degradation rates at 70% for Mmethylene blue (MB) and 85% for Congo red (CR). It is found that the genetic stability of 3H6 remains active for ten generations. The size of oxidase is 65 kDa, and optimum temperature for reaction is 30 °C with 4.5 pH. This study presents that the first combined mutagenesis approach by ARPT-UV on fungus species generates an impressive increment of acid dye oxidases production. As such, this method presents a cost-effective alternative to mitigate hazardous dye pollution.

Development of CRISPR-Cas13a-based antimicrobials capable of sequence-specific killing of target bacteria.

The emergence of antimicrobial-resistant bacteria is an increasingly serious threat to global health, necessitating the development of innovative antimicrobials. Here we report the development of a series of CRISPR-Cas13a-based antibacterial nucleocapsids, termed CapsidCas13a(s), capable of sequence-specific killing of carbapenem-resistant Escherichia coli and methicillin-resistant Staphylococcus aureus by recognizing corresponding antimicrobial resistance genes. CapsidCas13a constructs are generated by packaging programmed CRISPR-Cas13a into a bacteriophage capsid to target antimicrobial resistance genes. Contrary to Cas9-based antimicrobials that lack bacterial killing capacity when the target genes are located on a plasmid, the CapsidCas13a(s) exhibit strong bacterial killing activities upon recognizing target genes regardless of their location. Moreover, we also demonstrate that the CapsidCas13a(s) can be applied to detect bacterial genes through gene-specific depletion of bacteria without employing nucleic acid manipulation and optical visualization devices. Our data underscore the potential of CapsidCas13a(s) as both therapeutic agents against antimicrobial-resistant bacteria and nonchemical agents for detection of bacterial genes.

Corrigendum: Composition and Diversity of CRISPR-Cas13a Systems in the Genus Leptotrichia.

[This corrects the article DOI: 10.3389/fmicb.2019.02838.].

Complete Genome Sequence of the Methicillin-Resistant Staphylococcus aureus Strain JMUB3031, Isolated from a Patient with Fatal Community-Acquired Pneumonia.

Severe community-acquired pneumonia (CAP) caused by methicillin-resistant Staphylococcus aureus (MRSA) is relatively rare and is usually associated with rapid progression to death. Here, we report the complete genome sequence of the MRSA strain JMUB3031, which was isolated from a patient with fatal CAP.

Composition and Diversity of CRISPR-Cas13a Systems in the Genus Leptotrichia.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13a, previously known as CRISPR-C2c2, is the most recently identified RNA-guided RNA-targeting CRISPR-Cas system that has the unique characteristics of both targeted and collateral single-stranded RNA (ssRNA) cleavage activities. This system was first identified in Leptotrichia shahii. Here, the complete whole genome sequences of 11 Leptotrichia strains were determined and compared with 18 publicly available Leptotrichia genomes in regard to the composition, occurrence and diversity of the CRISPR-Cas13a, and other CRISPR-Cas systems. Various types of CRISPR-Cas systems were found to be unevenly distributed among the Leptotrichia genomes, including types I-B (10/29, 34.4%), II-C (1/29, 2.6%), III-A (6/29, 15.4%), III-D (6/29, 15.4%), III-like (3/29, 7.7%), and VI-A (11/29, 37.9%), while 8 strains (20.5%) had no CRISPR-Cas system at all. The Cas13a effectors were found to be highly divergent with amino acid sequence similarities ranging from 61% to 90% to that of L. shahii, but their collateral ssRNA cleavage activities leading to impediment of bacterial growth were conserved. CRISPR-Cas spacers represent a sequential achievement of former intruder encounters, and the retained spacers reflect the evolutionary phylogeny or relatedness of strains. Analysis of spacer contents and numbers among Leptotrichia species showed considerable diversity with only 4.4% of spacers (40/889) were shared by two strains. The organization and distribution of CRISPR-Cas systems (type I-VI) encoded by all registered Leptotrichia species revealed that effector or spacer sequences of the CRISPR-Cas systems were very divergent, and the prevalence of types I, III, and VI was almost equal. There was only one strain carrying type II, while none carried type IV or V. These results provide new insights into the characteristics and divergences of CRISPR-Cas systems among Leptotrichia species.

Complete genome sequencing of three human clinical isolates of Staphylococcus caprae reveals virulence factors similar to those of S. epidermidis and S. capitis.

BACKGROUND: Staphylococcus caprae is an animal-associated bacterium regarded as part of goats' microflora. Recently, S. caprae has been reported to cause human nosocomial infections such as bacteremia and bone and joint infections. However, the mechanisms responsible for the development of nosocomial infections remain largely unknown. Moreover, the complete genome sequence of S. caprae has not been determined. RESULTS: We determined the complete genome sequences of three methicillin-resistant S. caprae strains isolated from humans and compared these sequences with the genomes of S. epidermidis and S. capitis, both of which are closely related to S. caprae and are inhabitants of human skin capable of causing opportunistic infections. The genomes showed that S. caprae JMUB145, JMUB590, and JMUB898 strains contained circular chromosomes of 2,618,380, 2,629,173, and 2,598,513 bp, respectively. JMUB145 carried type V SCCmec, while JMUB590 and JMUB898 had type IVa SCCmec. A genome-wide phylogenetic SNP tree constructed using 83 complete genome sequences of 24 Staphylococcus species and 2 S. caprae draft genome sequences confirmed that S. caprae is most closely related to S. epidermidis and S. capitis. Comparative complete genome analysis of eight S. epidermidis, three S. capitis and three S. caprae strains revealed that they shared similar virulence factors represented by biofilm formation genes. These factors include wall teichoic acid synthesis genes, poly-gamma-DL-glutamic acid capsule synthesis genes, and other genes encoding nonproteinaceous adhesins. The 17 proteinases/adhesins and extracellular proteins known to be associated with biofilm formation in S. epidermidis were also conserved in these three species, and their biofilm formation could be detected in vitro. Moreover, two virulence-associated gene clusters, the type VII secretion system and capsular polysaccharide biosynthesis gene clusters, identified in S. aureus were present in S. caprae but not in S. epidermidis and S. capitis genomes. CONCLUSION: The complete genome sequences of three methicillin-resistant S. caprae isolates from humans were determined for the first time. Comparative genome analysis revealed that S. caprae is closely related to S. epidermidis and S. capitis at the species level, especially in the ability to form biofilms, which may lead to increased virulence during the development of S. caprae infections.

Selection of suitable reference genes for gene expression studies in Staphylococcus capitis during growth under erythromycin stress.

Accurate and reproducible measurement of gene transcription requires appropriate reference genes, which are stably expressed under different experimental conditions to provide normalization. Staphylococcus capitis is a human pathogen that produces biofilm under stress, such as imposed by antimicrobial agents. In this study, a set of five commonly used staphylococcal reference genes (gyrB, sodA, recA, tuf and rpoB) were systematically evaluated in two clinical isolates of Staphylococcus capitis (S. capitis subspecies urealyticus and capitis, respectively) under erythromycin stress in mid-log and stationary phases. Two public software programs (geNorm and NormFinder) and two manual calculation methods, reference residue normalization (RRN) and relative quantitative (RQ), were applied. The potential reference genes selected by the four algorithms were further validated by comparing the expression of a well-studied biofilm gene (icaA) with phenotypic biofilm formation in S. capitis under four different experimental conditions. The four methods differed considerably in their ability to predict the most suitable reference gene or gene combination for comparing icaA expression under different conditions. Under the conditions used here, the RQ method provided better selection of reference genes than the other three algorithms; however, this finding needs to be confirmed with a larger number of isolates. This study reinforces the need to assess the stability of reference genes for analysis of target gene expression under different conditions and the use of more than one algorithm in such studies. Although this work was conducted using a specific human pathogen, it emphasizes the importance of selecting suitable reference genes for accurate normalization of gene expression more generally.

Antibiotic regimen based on population analysis of residing persister cells eradicates Staphylococcus epidermidis biofilms.

Biofilm formation is a major pathogenicity strategy of Staphylococcus epidermidis causing various medical-device infections. Persister cells have been implicated in treatment failure of such infections. We sought to profile bacterial subpopulations residing in S. epidermidis biofilms, and to establish persister-targeting treatment strategies to eradicate biofilms. Population analysis was performed by challenging single biofilm cells with antibiotics at increasing concentrations ranging from planktonic minimum bactericidal concentrations (MBCs) to biofilm MBCs (MBCbiofilm). Two populations of "persister cells" were observed: bacteria that survived antibiotics at MBCbiofilm for 24/48 hours were referred to as dormant cells; those selected with antibiotics at 8 X MICs for 3 hours (excluding dormant cells) were defined as tolerant-but-killable (TBK) cells. Antibiotic regimens targeting dormant cells were tested in vitro for their efficacies in eradicating persister cells and intact biofilms. This study confirmed that there are at least three subpopulations within a S. epidermidis biofilm: normal cells, dormant cells, and TBK cells. Biofilms comprise more TBK cells and dormant cells than their log-planktonic counterparts. Using antibiotic regimens targeting dormant cells, i.e. effective antibiotics at MBCbiofilm for an extended period, might eradicate S. epidermidis biofilms. Potential uses for this strategy are in antibiotic lock techniques and inhaled aerosolized antibiotics.

Effects of erythromycin on the phenotypic and genotypic biofilm expression in two clinical Staphylococcus capitis subspecies and a functional analysis of Ica proteins in S. capitis.

The ica operon encoding polysaccharide intercellular adhesion, which facilitates biofilm formation in staphylococci, has been extensively studied in Staphylococcus epidermidis and Staphylococcus aureus. Based on in silico analysis, we suggest the following functional model for Ica proteins in S. capitis. IcaA is responsible for polysaccharide synthesis. IcaA and IcaD complete transferring the growing sugar chain to the cell surface; IcaB is a deacetylase, with the same function as IcaB of S. epidermidis. IcaC mainly modifies the synthesized glucan by acetylation. We also examined the effects of subinhibitory concentrations of erythromycin on phenotypic biofilm expression and transcription of biofilm-related genes, using isolates representing the two subspecies of Staphylococcus capitis and different biofilm and resistance phenotypes. On induction with erythromycin, biofilm density was strongly elevated in two erythromycin-resistant S. capitis, but not in three susceptible isolates. In the representative erythromycin-resistant S. capitis subsp. urealyticus, there were significant upregulations of the icaA gene and its positive regulator sarA on transition to the stationary phase without erythromycin induction. There were also significant increases in the transcription levels of icaA, rsbU and sigB corresponding to a very strong biofilm phenotype in the stationary phase on erythromycin stress. In contrast, the representative erythromycin-susceptible S. capitis subsp. capitis displayed upregulation only of altE on entry into the stationary phase with erythromycin induction, but this change was not associated with enhancement of biofilm production. These findings suggest that the two subspecies of S. capitis adopt different pathogenesis and survival strategies to adapt to a hostile environment.

Enhancing DNA electro-transformation efficiency on a clinical Staphylococcus capitis isolate.

Clinical staphylococcus isolates possess a stronger restriction-modification (RM) barrier than laboratory strains. Clinical isolates are therefore more resistant to acceptance of foreign genetic material than laboratory strains, as their restriction systems more readily recognize and destroy foreign DNA. This stronger barrier consequently restricts genetic studies to a small number of domestic strains that are capable of accepting foreign DNA. In this study, an isolate of Staphylococcus capitis, obtained from the blood of a very low birth-weight baby, was transformed with a shuttle vector, pBT2. Optimal conditions for electro-transformation were as follows: cells were harvested at mid-log phase, electro-competent cells were prepared; cells were pre-treated at 55°C for 1min; 3µg of plasmid DNA was mixed with 70-80µL of competent cells (3-4×10(10)cells/mL) at 20°C in 0.5M sucrose, 10% glycerol; and electroporation was conducted using 2.1kV/cm field strength with a 0.1cm gap. Compared to the conventional method, which involves DNA electroporation of Staphylococcus aureus RN4220 as an intermediate strain to overcome the restriction barrier, our proposed approach exhibits a higher level (3 log10 units) of transformation efficiency. Heat treatment was used to temporarily inactivate the recipient RM barrier. Other important parameters contributing to improved electro-transformation efficiency were growth stage for cell harvesting, the quantity of DNA, the transformation temperature and field strength. The approach described here may facilitate genetic manipulations of this opportunistic pathogen.

Differences between two clinical Staphylococcus capitis subspecies as revealed by biofilm, antibiotic resistance, and pulsed-field gel electrophoresis profiling.

Coagulase-negative staphylococci have been identified as major causes of late-onset neonatal bacteremia in neonatal intensive care units. Sixty isolates of Staphylococcus capitis obtained from blood cultures of neonates between 2000 and 2005 were examined in this study. Biochemical analysis confirmed that 52 of these isolates belonged to the subsp. urealyticus, and the remaining 8 belonged to the subsp. capitis. Isolates of the predominant subsp. urealyticus clones were characterized by their resistance to penicillin, erythromycin, and oxacillin and their biofilm formation ability, whereas subsp. capitis isolates were generally antibiotic susceptible and biofilm negative. Pulsed-field gel electrophoresis (PFGE) after SacII digestion separated the 60 isolates into five major clusters. Sequence analysis showed that, in S. capitis, the ica operon plus the negative regulator icaR was 4,160 bp in length. PCRs demonstrated the presence of the ica operon in all isolates. Further analysis of five isolates (two biofilm-positive subsp. urealyticus, one biofilm-negative subsp. urealyticus, and two biofilm-negative subsp. capitis) revealed that the ica operons were identical in all of the biofilm-positive subsp. urealyticus strains; however, the biofilm-negative isolates showed variations. The distinctive phenotypic and genotypic characteristics revealed by this study may affect the epidemiology of the two subspecies of S. capitis in the clinical setting. These results may provide a better understanding of the contribution of these two species to bloodstream infections in neonates.