Numerical Study of Flow and Heat Transfer Characteristics for Al2O3 Nanofluid in a Double-Pipe Helical Coil Heat Exchanger.

To numerically investigate the flow and heat transfer characteristics of a water/Al2O3 nanofluid in a double-pipe helical coil heat exchanger, we simulated a two-phase Eulerian model to predict the heat transfer coefficient, Nusselt number, and pressure drop at various concentrations (i.e., volume fraction) and under diverse flow rates at the steady state. In this simulation, we used the k-epsilon turbulence model with an enhanced wall treatment method. The performance factor of the nanofluid was evaluated by accounting for the heat transfer and pressure drop characteristics. As a result, the heat transfer was enhanced by increasing the nanofluid concentration. The 1.0 vol.% nanofluid (i.e., the highest concentration) showed a heat transfer coefficient 1.43 times greater than water and a Nusselt number of 1.38 times greater than water. The pressure drop of nanofluids was greater than that of water due to the increased density and viscosity induced using nanoparticles. Based on the relationship between the Nusselt number and pressure drop, the 1.0 vol.% nanofluid was calculated to have a performance factor of 1.4 relative to water, indicating that the enhancement rate in heat transfer performance was greater than that in the pressure drop. In conclusion, the Al2O3 nanofluid shows potential as an enhanced working fluid in diverse heat transfer applications.

Characterization of thermostable bacteriophage CPD2 and its endolysin LysCPD2 as biocontrol agents against Clostridium perfringens.

Clostridium perfringens is one of the major foodborne pathogens in humans and animals. With the prevalence of antibiotic-resistant C. perfringens strains, bacteriophages and their endolysins have received considerable attention as promising alternatives to antibiotics. In this study, C. perfringens phage CPD2 was isolated from retail chicken samples. CPD2 belongs to the Podoviridae family and exhibits remarkable thermostability. While CPD2 has narrow host specificity, its endolysin LysCPD2 showed a broader lytic range, killing not only C. perfringens strains but other Gram-positive bacteria, such as B. cereus and B. subtilis. In addition, due to its exceptional thermal stability, LysCPD2 showed significant antibacterial ability against germinating C. perfringens spores during the heat activation process (75 °C for 20 min). Taken together, these results indicate that both thermostable phage CPD2 and its endolysin LysCPD2 can be used as efficient antimicrobial agents to control C. perfringens during thermal processing of foods.

LysGR1, a novel thermostable endolysin from Geobacillus stearothermophilus bacteriophage GR1.

Geobacillus stearothermophilus is a highly thermophilic, spore-forming Gram-positive bacterium that causes flat sour spoilage in low-acid canned foods. To address this problem, we isolated G. stearothermophilus-infecting phage GR1 from the soil and characterized its endolysin LysGR1. Phage GR1 belongs to the Siphoviridae family and possesses a genome of 79,387 DNA bps with 108 putative open reading frames. GR1 demonstrated a very low degree of homology to previously reported phages, indicating that it is novel. The endolysin of GR1 (LysGR1) contains an N-terminal amidase domain as an enzymatically active domain (EAD) and two C-terminal LysM domains as a cell wall binding domain (CBD). Although GR1 is specific to certain strains of G. stearothermophilus, LysGR1 showed a much broader lytic range, killing all the tested strains of G. stearothermophilus and several foodborne pathogens, such as Clostridium perfringens, Listeria monocytogenes, and Escherichia coli O157:H7. LysGR1_EAD, alone, also exhibits lytic activity against a wide range of bacteria, including Bacillus cereus, which is not terminated by a full-length endolysin. Both LysGR1 and its EAD effectively remove the G. stearothermophilus biofilms and are highly thermostable, retaining about 70% of their lytic activity after a 15-min incubation at 70°C. Considering the high thermal stability, broad lytic activity, and biofilm reduction efficacy of LysGR1 and its EAD, we hypothesize that these enzymes could act as promising biocontrol agents against G. stearothermophilus and as foodborne pathogens.

Isolation and Characterization of a Weizmannia coagulans Bacteriophage Youna2 and Its Endolysin PlyYouna2.

Weizmannia coagulans (formerly Bacillus coagulans) is Gram-positive, and spore-forming bacteria causing food spoilage, especially in acidic canned food products. To control W. coagulans, we isolated a bacteriophage Youna2 from a sewage sludge sample. Morphological analysis revealed that phage Youna2 belongs to the Siphoviridae family with a non-contractile and flexible tail. Youna2 has 52,903 bp double-stranded DNA containing 61 open reading frames. There are no lysogeny-related genes, suggesting that Youna2 is a virulent phage. plyYouna2, a putative endolysin gene was identified in the genome of Youna2 and predicted to be composed of a N-acetylmuramoyl-L-alanine amidase domain (PF01520) at the N-terminus and unknown function DUF5776 domain (PF19087) at the C-terminus. While phage Youna2 has a narrow host range, infecting only certain strains of W. coagulans, PlyYouna2 exhibited a broad antimicrobial spectrum beyond the Bacillus genus. Interestingly, PlyYouna2 can lyse Gram-negative bacteria such as Escherichia coli, Yersinia enterocolitica, Pseudomonas putida and Cronobacter sakazakii without other additives to destabilize bacterial outer membrane. To the best of our knowledge, Youna2 is the first W. coagulans-infecting phage and we speculate its endolysin PlyYouna2 can provide the basis for the development of a novel biocontrol agent against various foodborne pathogens.

Cell-specific cargo delivery using synthetic bacterial spores.

Delivery of cancer therapeutics to non-specific sites decreases treatment efficacy while increasing toxicity. In ovarian cancer, overexpression of the cell surface marker HER2, which several therapeutics target, relates to poor prognosis. We recently reported the assembly of biocompatible bacterial spore-like particles, termed "SSHELs." Here, we modify SSHELs with an affibody directed against HER2 and load them with the chemotherapeutic agent doxorubicin. Drug-loaded SSHELs reduce tumor growth and increase survival with lower toxicity in a mouse tumor xenograft model compared with free drug and with liposomal doxorubicin by preferentially accumulating in the tumor mass. Target cells actively internalize and then traffic bound SSHELs to acidic compartments, whereupon the cargo is released to the cytosol in a pH-dependent manner. We propose that SSHELs represent a versatile strategy for targeted drug delivery, especially in cancer settings.

Complete Genome Sequence of Levilactobacillus brevis Bacteriophage ENFP1.

We describe the complete genome sequence of bacteriophage ENFP1, which infects Levilactobacillus brevis; it has a capsid width of 83 nm and a tail length of 144 nm. The 138.6-kb genome, containing 190 predicted protein-coding genes, is similar (88.03% nucleotide sequence identity) to that of L. brevis phage 521B.

An Antibacterial Nanorobotic Approach for the Specific Targeting and Removal of Multiple Drug-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) causes diseases ranging from skin infections to lethal sepsis and has become a serious threat to human health due to multiple-drug resistance (MDR). Therefore, a resistance-free antibacterial therapy is necessary to overcome MDR MRSA infections. In this study, an antibacterial nanorobot (Ab-nanobot) is developed wherein a cell wall-binding domain (CBD)-endolysin, acting as a sensor, is covalently conjugated with an actuator consisting of an iron oxide/silica core-shell. The CBD-endolysin sensor shows an excellent specificity to detect, bind, and accumulate on the S. aureus USA300 cell surface even in a bacterial consortium, and in host cell infections. Ab-nanobot specifically captures and kills MRSA in response to medically approved radiofrequency (RF) electromagnetic stimulation (EMS) signal. When Ab-nanobot receives the RF-EMS signal on the cell surface, actuator induces cell death in MRSA with 99.999% removal within 20 min by cell-wall damage via generation of localized heat and reactive oxygen species. The in vivo efficacy of Ab-nanobot is proven using a mice subcutaneous skin infection model. Collectively, this study offers a nanomedical resistance-free strategy to overcome MDR MRSA infections by providing a highly specific nanorobot for S. aureus.

Simultaneous Control of Staphylococcus aureus and Bacillus cereus Using a Hybrid Endolysin LysB4EAD-LysSA11.

Bacteriophage endolysins have attracted attention as promising alternatives to antibiotics, and their modular structure facilitates endolysin engineering to develop novel endolysins with enhanced versatility. Here, we constructed hybrid proteins consisting of two different endolysins for simultaneous control of two critical foodborne pathogens, Staphylococcus aureus and Bacillus cereus. The full-length or enzymatically active domain (EAD) of LysB4, an endolysin from the B. cereus-infecting phage B4, was fused to LysSA11, an endolysin of the S. aureus-infecting phage SA11, via a helical linker in both orientations. The hybrid proteins maintained the lytic activity of their parental endolysins against both S. aureus and B. cereus, but they showed an extended antimicrobial spectrum. Among them, the EAD of LysB4 fused with LysSA11 (LysB4EAD-LyaSA11) showed significantly increased thermal stability compared to its parental endolysins. LysB4EAD-LysSA11 exhibited high lytic activity at pH 8.0-9.0 against S. aureus and at pH 5.0-10.0 against B. cereus, but the lytic activity of the protein decreased in the presence of NaCl. In boiled rice, treatment with 3.0 µM of LysB4EAD-LysSA11 reduced the number of S. aureus and B. cereus to undetectable levels within 2 h and also showed superior antimicrobial activity to LyB4EAD and LysSA11 in combination. These results suggest that LysB4EAD-LysSA11 could be a potent antimicrobial agent for simultaneous control of S. aureus and B. cereus.

Development of a Novel Chimeric Endolysin, Lys109 With Enhanced Lytic Activity Against Staphylococcus aureus.

As the incidence of antibiotic-resistant bacteria has become increased, phage endolysins are believed as one of the promising alternatives to antibiotics. However, the discovery of potent endolysin is still challenging because it is labor intensive and difficult to obtain a soluble form with high lytic activity. In this respect, the modular structures of Gram-positive endolysins can provide an opportunity to develop novel endolysins by domain rearrangement. In this study, a random domain swapping library of four different endolysins from phages infecting Staphylococcus aureus was constructed and screened to obtain engineered endolysins. The novel chimeric endolysin, Lys109 was selected and characterized for its staphylolytic activity. Lys109 exhibited greater bacterial cell lytic activity than its parental endolysins against staphylococcal planktonic cells and biofilms, showing highly improved activity in eliminating S. aureus from milk and on the surface of stainless steel. These results demonstrate that a novel chimeric endolysin with higher activity and solubility can be developed by random domain swapping and that this chimeric endolysin has a great potential as an antimicrobial agent.

Structural Basis for Cell-Wall Recognition by Bacteriophage PBC5 Endolysin.

Phage endolysins are hydrolytic enzymes that cleave the bacterial cell wall during the lytic cycle. We isolated the bacteriophage PBC5 against Bacillus cereus, a major foodborne pathogen, and describe the molecular interaction between endolysin LysPBC5 and the host peptidoglycan structure. LysPBC5 has an N-terminal glycoside hydrolase 25 domain, and a C-terminal cell-wall binding domain (CBD) that is critical for specific cell-wall recognition and lysis. The crystal and solution structures of CBDs reveal tandem SH3b domains that are tightly engaged with each other. The CBD binds to the peptidoglycan in a bidentate manner via distal ß sheet motifs with pseudo 2-fold symmetry, which can explain its high affinity and host specificity. The CBD primarily interacts with the glycan strand of the peptidoglycan layer instead of the peptide crosslink, implicating the tertiary structure of peptidoglycan as the recognition motif of endolysins.

LysPBC2, a Novel Endolysin Harboring a Bacillus cereus Spore Binding Domain.

To control the spore-forming human pathogen Bacillus cereus, we isolated and characterized a novel endolysin, LysPBC2, from a newly isolated B. cereus phage, PBC2. Compared to the narrow host range of phage PBC2, LysPBC2 showed very broad lytic activity against all Bacillus, Listeria, and Clostridium species tested. In addition to a catalytic domain and a cell wall binding domain, LysPBC2 has a spore binding domain (SBD) partially overlapping its catalytic domain, which specifically binds to B. cereus spores but not to vegetative cells of B. cereus Both immunogold electron microscopy and a binding assay indicated that the SBD binds the external region of the spore cortex layer. Several amino acid residues required for catalytic or spore binding activity of LysPBC2 were determined by mutagenesis studies. Interestingly, LysPBC2 derivatives with impaired spore binding activity showed an increased lytic activity against vegetative cells of B. cereus compared with that of wild-type LysPBC2. Further biochemical studies revealed that these LysPBC2 derivatives have lower thermal stability, suggesting a stabilizing role of SBD in LysPBC2 structure.IMPORTANCE Bacteriophages produce highly evolved lytic enzymes, called endolysins, to lyse peptidoglycan and release their progeny from bacterial cells. Due to their potent lytic activity and specificity, the use of endolysins has gained increasing attention as a natural alternative to antibiotics. Since most endolysins from Gram-positive-bacterium-infecting phages have a modular structure, understanding the function of each domain is crucial to make effective endolysin-based therapeutics. Here, we report the functional and biochemical characterization of a Bacillus cereus phage endolysin, LysPBC2, which has an unusual spore binding domain and a cell wall binding domain. A single point mutation in the spore binding domain greatly enhanced the lytic activity of endolysin at the cost of reduced thermostability. This work contributes to the understanding of the role of each domain in LysPBC2 and will provide insight for the rational design of efficient antimicrobials or diagnostic tools for controlling B. cereus.

Smart Gold Nanoparticle-Stabilized Ultrasound Microbubbles as Cancer Theranostics.

Smart gold nanoparticle-stabilized microbubbles (SAuMBs) composed of a gas-filled core and shell including smart gold nanoparticles (SAuNPs) which can be aggregated in tumors were applied as ultrasound-mediated cancer theranostics. The gas core in the microstructure enabled the detection of tumors using ultrasound and facilitated the delivery of SAuNPs by sonoporation. The SAuNPs spontaneously aggregated in tumors, which allowed photoacoustic (PA) monitoring and photothermal treatment (PTT) of tumors.

Vaccine display on artificial bacterial spores enhances protective efficacy against Staphylococcus aureus infection.

Spores of Bacillus subtilis are encased in a protein coat composed of ∼80 different proteins. Recently, we reconstituted the basement layer of the coat, composed of two structural proteins (SpoVM and SpoIVA) around spore-sized silica beads encased in a lipid bilayer, to create synthetic spore-like particles termed 'SSHELs'. We demonstrated that SSHELs could display thousands of copies of proteins and small molecules of interest covalently linked to SpoIVA. In this study, we investigated the efficacy of SSHELs in delivering vaccines. We show that intramuscular vaccination of mice with undecorated one micron-diameter SSHELs elicited an antibody response against SpoIVA. We further demonstrate that SSHELs covalently modified with a catalytically inactivated staphylococcal alpha toxin variant (HlaH35L), without an adjuvant, resulted in improved protection against Staphylococcus aureus infection in a bacteremia model as compared to vaccination with the antigen alone. Although vaccination with either HlaH35L or HlaH35L conjugated to SSHELs similarly elicited the production of neutralizing antibodies to Hla, we found that a subset of memory T cells was differentially activated when the antigen was delivered on SSHELs. We propose that the particulate nature of SSHELs elicits a more robust immune response to the vaccine that results in superior protection against subsequent S. aureus infection.

The Auxiliary Role of the Amidase Domain in Cell Wall Binding and Exolytic Activity of Staphylococcal Phage Endolysins.

In response to increasing concern over antibiotic-resistant Staphylococcus aureus, the development of novel antimicrobials has been called for, with bacteriophage endolysins having received considerable attention as alternatives to antibiotics. Most staphylococcal phage endolysins have a modular structure consisting of an N-terminal cysteine, histidine-dependent amidohydrolases/peptidase domain (CHAP), a central amidase domain, and a C-terminal cell wall binding domain (CBD). Despite extensive studies using truncated staphylococcal endolysins, the precise function of the amidase domain has not been determined. Here, a functional analysis of each domain of two S. aureus phage endolysins (LysSA12 and LysSA97) revealed that the CHAP domain conferred the main catalytic activity, while the central amidase domain showed no enzymatic activity in degrading the intact S. aureus cell wall. However, the amidase-lacking endolysins had reduced hydrolytic activity compared to the full-length endolysins. Comparison of the binding affinities of fusion proteins consisting of the green fluorescent protein (GFP) with CBD and GFP with the amidase domain and CBD revealed that the major function of the amidase domain was to enhance the binding affinity of CBD, resulting in higher lytic activity of endolysin. These results suggest an auxiliary binding role of the amidase domain of staphylococcal endolysins, which can be useful information for designing effective antimicrobial and diagnostic agents against S. aureus.

Multiplexed Detection of Foodborne Pathogens from Contaminated Lettuces Using a Handheld Multistep Lateral Flow Assay Device.

This paper presents a handheld device that is capable of simplifying multistep assays to perform sensitive detection of foodborne pathogens. The device is capable of multiplexed detection of Escherichia coli (E. coli) O157:H7, Salmonella Typhimurium (S. Typhimurium), Staphylococcus aureus, and Bacillus cereus. The limit of detection for each bacterium was characterized, and then, the detection of bacteria from contaminated fresh lettuces was demonstrated for two representative foodborne pathogens. We employed a sample pretreatment protocol to recover and concentrate target bacteria from contaminated lettuces, which can detect 1.87 × 104 CFU of E. coli O157:H7 and 1.47 × 104 CFU of S. Typhimurium/1 g of lettuce without an enrichment process. Lastly, we demonstrated that the limit of detection can be reduced to 1 CFU of E. coli O157:H7 and 1 CFU of S. Typhimurium/1 g of lettuce by including a 6 h enrichment of contaminated lettuces in growth media before pretreatment.

Lateral flow assay-based bacterial detection using engineered cell wall binding domains of a phage endolysin.

The development of a cost-effective and efficient bacterial detection assay is essential for diagnostic fields, particularly in resource-poor settings. Although antibodies have been widely used for bacterial capture, the production of soluble antibodies is still expensive and time-consuming. Here, we developed a nitrocellulose-based lateral flow assay using cell wall binding domains (CBDs) from phage as a recognition element and colloidal gold nanoparticles as a colorimetric signal for the detection of a model pathogenic bacterium, Bacillus cereus (B. cereus). To improve conjugation efficiency and detection sensitivity, cysteine-glutathione-S-transferase-tagged CBDs and maltose-binding protein-tagged CBDs were produced in Escherichia coli (E. coli) and incorporated in our assays. The sensitivity of the strip to detect B. cereus was 1×104 CFU/mL and the overall assay time was 20min. The assay showed superior results compared to the antibody-based approach, and did not show any significant cross-reactivity. This proof of concept study indicates that the lateral flow assay using engineered CBDs hold considerable promise as simple, rapid, and cost-effective biosensors for whole cell detection.

Characterization of LysPBC4, a novel Bacillus cereus-specific endolysin of bacteriophage PBC4.

Bacillus cereus is a spore-forming, Gram-positive bacterium and is a major food-borne pathogen. A B. cereus-specific bacteriophage PBC4 was isolated from the soil of a stock farm, and its genome was analyzed. PBC4 belongs to the Siphoviridae family and has a genome consisting of 80 647-bp-long double-stranded DNA, including 123 genes and two tRNAs. LysPBC4, the endolysin of PBC4, has an enzymatically active domain (EAD) on its N-terminal region and a putative cell wall-binding domain (CBD) on its C-terminal region, respectively. Although the phage PBC4 showed a very limited host range, LysPBC4 could lyse all of the B. cereus strains tested. However, LysPBC4 did not kill other bacteria such as B. subtilis or Listeria, indicating that the endolysin has specific lytic activity against the B. cereus group species. Furthermore, LysPBC4_CBD fused with enhanced green fluorescent protein (EGFP) could decorate limited strains of B. cereus group, suggesting that the LysPBC4_CBD may be a promising material for specific detection of B. cereus.

Identification of a Bacteria-Specific Binding Protein from the Sequenced Bacterial Genome.

Novel and specific recognition elements are of central importance in the development of a pathogen detection method. Here, we describe a simple method for identifying the cell-wall binding domain (CBD) from a sequenced bacterial genome employing homology search for phage lysin genes. A putative CBD (CPF369_CBD) was identified from a genome of Clostridium perfringens type strain ATCC 13124, and its function was studied with the CBDGFP fusion protein recombinantly expressed in Escherichia coli. Fluorescence microscopy showed the specific binding of the fusion protein to C. perfringens cells, which demonstrates the potential of this method for the identification of novel bioprobes for specific detection of pathogenic bacteria.

A novel and highly specific phage endolysin cell wall binding domain for detection of Bacillus cereus.

Rapid, specific and sensitive detection of pathogenic bacteria is crucial for public health and safety. Bacillus cereus is harmful as it causes foodborne illness and a number of systemic and local infections. We report a novel phage endolysin cell wall-binding domain (CBD) for B. cereus and the development of a highly specific and sensitive surface plasmon resonance (SPR)-based B. cereus detection method using the CBD. The newly discovered CBD from endolysin of PBC1, a B. cereus-specific bacteriophage, provides high specificity and binding capacity to B. cereus. By using the CBD-modified SPR chips, B. cereus can be detected at the range of 10(5)-10(8) CFU/ml. More importantly, the detection limit can be improved to 10(2) CFU/ml by using a subtractive inhibition assay based on the pre-incubation of B. cereus and CBDs, removal of CBD-bound B. cereus, and SPR detection of the unbound CBDs. The present study suggests that the small and genetically engineered CBDs can be promising biological probes for B. cereus. We anticipate that the CBD-based SPR-sensing methods will be useful for the sensitive, selective, and rapid detection of B. cereus.

Bacteriophage PBC1 and its endolysin as an antimicrobial agent against Bacillus cereus.

Bacillus cereus is an opportunistic human pathogen responsible for food poisoning and other, nongastrointestinal infections. Due to the emergence of multidrug-resistant B. cereus strains, the demand for alternative therapeutic options is increasing. To address these problems, we isolated and characterized a Siphoviridae virulent phage, PBC1, and its lytic enzymes. PBC1 showed a very narrow host range, infecting only 1 of 22 B. cereus strains. Phylogenetic analysis based on the major capsid protein revealed that PBC1 is more closely related to the Bacillus clarkii phage BCJA1c and phages of lactic acid bacteria than to the phages infecting B. cereus. Whole-genome comparison showed that the late-gene region, including the terminase gene, structural genes, and holin gene of PBC1, is similar to that from B. cereus temperate phage 250, whereas their endolysins are different. Compared to the extreme host specificity of PBC1, its endolysin, LysPBC1, showed a much broader lytic spectrum, albeit limited to the genus Bacillus. The catalytic domain of LysPBC1 when expressed alone also showed Bacillus-specific lytic activity, which was lower against the B. cereus group but higher against the Bacillus subtilis group than the full-length protein. Taken together, these results suggest that the virulent phage PBC1 is a useful component of a phage cocktail to control B. cereus, even with its exceptionally narrow host range, as it can kill a strain of B. cereus that is not killed by other phages, and that LysPBC1 is an alternative biocontrol agent against B. cereus.