Therapeutic Effect of a Newly Isolated Lytic Bacteriophage against Multi-Drug-Resistant Cutibacterium acnes Infection in Mice.

Acne vulgaris, which is mostly associated with the colonization of Cutibacterium acnes (C. acnes), is a common skin inflammatory disease in teenagers. However, over the past few years, the disease has extended beyond childhood to chronically infect approximately 40% of adults. While antibiotics have been used for several decades to treat acne lesions, antibiotic resistance is a growing crisis; thus, finding a new therapeutic target is urgently needed. Studies have shown that phage therapy may be one alternative for treating multi-drug-resistant bacterial infections. In the present study, we successfully isolated a C. acnes phage named TCUCAP1 from the skin of healthy volunteers. Morphological analysis revealed that TCUCAP1 belongs to the family Siphoviridae with an icosahedral head and a non-contractile tail. Genome analysis found that TCUCAP1 is composed of 29,547 bp with a G+C content of 53.83% and 56 predicted open reading frames (ORFs). The ORFs were associated with phage structure, packing, host lysis, DNA metabolism, and additional functions. Phage treatments applied to mice with multi-drug-resistant (MDR) C.-acnes-induced skin inflammation resulted in a significant decrease in inflammatory lesions. In addition, our attempt to formulate the phage into hydroxyethyl cellulose (HEC) cream may provide new antibacterial preparations for human infections. Our results demonstrate that TCUCAP1 displays several features that make it an ideal candidate for the control of C. acnes infections.

Identification of carbonylated proteins in a bactericidal process induced by curcumin with blue light irradiation on imipenem-resistant Acinetobacter baumannii.

RATIONALE: Antimicrobial photodynamic treatment is potentially an alternative to antibiotics and is also effective against viruses, fungi and some cancers. Our previous studies have shown that blue light combined with curcumin, a chemical from the turmeric plant, exerted effective antimicrobial activity via photodynamic treatment. The study reported in this paper investigates which target proteins are affected after the treatment. METHODS: We treated imipenem-resistant Acinetobacter baumannii with blue light and curcumin and used protein carbonylation as a marker for oxidative damage. After treatment, the bacterial proteins were extracted and the protein carbonyls marked using dinitrophenylhydrazide. After enzyme digestion, we used liquid chromatography/nano-electrospray ionization (LC/nano-ESI) ion trap mass spectrometry to identify bacterial peptides from a customized database. The functional enrichment analyses of the identified proteins were performed using gene ontology annotation and the STRING protein-protein interaction network. RESULTS: The application of curcumin with blue light showed good antibacterial activity against imipenem-resistant A. baumannii. Using a shotgun proteomics approach, the carbonylated proteins in A. baumannii caused by the photolytic curcumin were identified. The results showed that the proteins related to membrane structures, translation and response to oxidative stress were preferentially modified. CONCLUSIONS: The photolytic curcumin treatment could be a potential alternative to antibiotics for bacterial infection. In this study, the shotgun proteomics strategy allows us to explore the possible bactericidal mechanisms under this oxidative stress. The result provides a reference for future studies on the enhancement of the action of photolytic curcumin.

Antimycobacterial Activities of Endolysins Derived From a Mycobacteriophage, BTCU-1.

The high incidence of Mycobacterium infection, notably multidrug-resistant M. tuberculosis infection, has become a significant public health concern worldwide. In this study, we isolate and analyze a mycobacteriophage, BTCU-1, and a foundational study was performed to evaluate the antimycobacterial activity of BTCU-1 and its cloned lytic endolysins. Using Mycobacterium smegmatis as host, a mycobacteriophage, BTCU-1, was isolated from soil in eastern Taiwan. The electron microscopy images revealed that BTCU-1 displayed morphology resembling the Siphoviridae family. In the genome of BTCU-1, two putative lytic genes, BTCU-1_ORF7 and BTCU-1_ORF8 (termed lysA and lysB, respectively), were identified, and further subcloned and expressed in Escherichia coli. When applied exogenously, both LysA and LysB were active against M. smegmatis tested. Scanning electron microscopy revealed that LysA and LysB caused a remarkable modification of the cell shape of M. smegmatis. Intracellular bactericidal activity assay showed that treatment of M. smegmatis-infected RAW 264.7 macrophages with LysA or LysB resulted in a significant reduction in the number of viable intracellular bacilli. These results indicate that the endolysins derived from BTCU-1 have antimycobacterial activity, and suggest that they are good candidates for therapeutic/disinfectant agents to control mycobacterial infections.

Diagnosis of ß-lactam resistance in Acinetobacter baumannii using shotgun proteomics and LC-nano-electrospray ionization ion trap mass spectrometry.

Acinetobacter baumannii is an important nosocomial pathogen that often affects critically ill patients in intensive care units. ß-Lactam antibiotics are the most commonly prescribed drugs for infectious diseases caused by A. baumannii. Our aim is to develop an accurate and rapid shotgun proteomics method for the identification of ß-lactam-resistant A. baumannii pathogens. In the present study, we used automated data-dependent scanning on a nano-LC/ion trap mass spectrometer to characterize proteotypic peptides of A. baumannii. Then, we used SEQUEST software to search specific databases, the ß-lactam-resistance protein database of A. baumannii (BRPDAB). We successfully found a number of associated antibiotic-resistant proteins, including AmpC, ß-lactamase, and carO, in clinical resistant strains of A. baumannii and differentiated them from wild-type A. baumannii strains. We used the results of the search to identify A. baumannii pathogens and found a ß-lactam-resistant clinical strain of A. baumannii using Uniprot annotations, Gene Ontology (GO), and BLAST bioinformatics tools. This proteomic study will provide a platform for the rapid diagnosis of wild-type and resistant strains of A. baumannii, which would be useful for the medical treatment of these strains.

Potential of bacteriophage ΦAB2 as an environmental biocontrol agent for the control of multidrug-resistant Acinetobacter baumannii.

BACKGROUND: Multidrug-resistant Acinetobacter baumannii (MDRAB) is associated with nosocomial infections worldwide. To date, the use of a phage to prevent infections caused by MDRAB has not been demonstrated. RESULTS: The MDRAB-specific phage ϕAB2 was stable at 4°C and pH 7 in 0.5% chloroform solution, and showed a slight decrease in plaque-forming units (PFU)/ml of 0.3-0.9 log after 330 days of storage. The addition of ϕAB2 at a concentration of at least 105 PFU/ml to an A. baumannii M3237 suspension killed >99.9% of A. baumannii M3237 after 5 min, regardless of A. baumannii M3237 concentration (104, 105, or 106 colony-forming units (CFU)/ml). The addition of ϕAB2 at a concentration of 108 PFU/slide (>107 PFU/cm²) to glass slides containing A. baumannii M3237 at 104, 105, or 106 CFU/slide, significantly reduced bacterial numbers by 93%, 97%, and 99%, respectively. Thus, this concentration is recommended for decontamination of glass surfaces. Moreover, infusion of ϕAB2 into 10% glycerol exhibited strong anti-MDRAB activity (99.9% reduction), even after 90 days of storage. Treatment of a 10% paraffin oil-based lotion with ϕAB2 significantly reduced (99%) A. baumannii M3237 after 1 day of storage. However, ϕAB2 had no activity in the lotion after 1 month of storage. CONCLUSIONS: Phages may be useful for reducing MDRAB contamination in liquid suspensions or on hard surfaces. Phages may also be inoculated into a solution to produce an antiseptic hand wash. However, the phage concentration and incubation time (the duration of phage contact with bacteria) should be carefully considered to reduce the risk of MDRAB contamination.

Isolation and characterization of bacteriophages with activities against multi-drug-resistant Acinetobacter nosocomialis causing bloodstream infection in vivo.

BACKGROUND: Acinetobacter nosocomialis (A. nosocomialis) is a glucose non-fermentative, gram-negative bacillus that belongs to the Acinetobacter calcoaceticus-baumannii complex. In recent years, studies have found an increased clinical prevalence of A. nosocomialis. However, given the increasing trend of antibiotic resistance, developing new antibacterial agents is vital. Currently, research regarding bacteriophage therapy against A. nosocomialis is only limited. METHODS: Two A. nosocomialis bacteriophages, TCUAN1 and TCUAN2, were isolated from sewage. Experiments such as transmission electron microscopy (TEM), host-range analysis, and sequencing were performed to determine their biological and genomic characteristics. TCUAN2 were further subjected to in vivo experiments and their derived-endolysin were cloned and tested against their bacteria host. RESULTS: Transmission electron microscopy revealed that TCUAN1 and TCUAN2 belong to Myoviridae and Podoviridae, respectively. Both phages show a broad host spectrum and rapid adsorption efficiency. Further biological analysis showed that TCUAN2 possesses a shorter latent period and larger burst size compared to TCUAN1. Because TCUAN2 showed a better antibacterial activity, it was injected into A. nosocomialis-infected mice which resulted in a significant decrease in bacterial load levels in the blood and increased the mice's survival. Finally, genomic analysis revealed that the complete nucleotide sequence of TCUAN1 is 49, 691 bps (containing 75 open reading frames) with a G + C content of 39.3%; whereas the complete nucleotide sequence of TCUAN2 is 41, 815 bps (containing 68 open reading frames) with a G + C content of 39.1%. The endolysin gene cloned and purified from TCUAN2 also showed antibacterial activity when used with a chelator EDTA.

Genomic analysis of bacteriophage ϕAB1, a ϕKMV-like virus infecting multidrug-resistant Acinetobacter baumannii.

We present the complete genomic sequence of a lytic bacteriophage ϕAB1 which can infect many clinical isolates of multidrug-resistant Acinetobacter baumannii. The recently isolated bacteriophage displays morphology resembling Podoviridae family. The ϕAB1 genome is a linear double-stranded DNA of 41,526 bp containing 46 possible open reading frames (ORFs). The majority of the predicted structural proteins were identified as part of the phage particle by mass spectrometry analysis. According to the virion morphology, overall genomic structure, and the phylogenetic tree of RNA polymerase, we propose that ϕAB1 is a new member of the ϕKMV-like phages. Additionally, we identified four ORFs encoding putative HNH endonucleases, one of which is presumed to integrate and create a genes-in-pieces DNA polymerase. Also, a potential lysis cassette was identified in the late genome. The lytic power of this bacteriophage combined with its specificity for A. baumannii makes ϕAB1 an attractive agent for therapeutic or disinfection applications.

Effect of Size and Concentration of Copper Nanoparticles on the Antimicrobial Activity in Escherichia coli through Multiple Mechanisms.

Metal and metal oxide nanoparticles, including copper nanoparticles (CuNPs), display antimicrobial activities and are regarded as promising microorganism inhibitors. Here, we explored the antimicrobial activity of CuNPs in Escherichia coli (E. coli) using two particle sizes (20 and 60 nm) and five concentrations (1, 5, 10, 50 and 100 µg/mL). The result showed a concentration-dependent trend of bactericidal activities for both size groups, with 20 nm particles more effective than 60 nm particles at low concentrations. The membrane disruption caused by CuNPs was confirmed by electron microscopy, PI staining and protein leaking analysis. However, the results of reactive oxygen species generation and genomic DNA damage revealed that the size and concentration of CuNPs were factors affecting the induction of multiple bactericidal mechanisms simultaneously on different scales. Further results of annexin V-PI staining supported this hypothesis by showing the shifting composition of the early-, late- and non-apoptotic dead cells across the CuNP groups. Many CuNP treatment groups were rescued when four mammalian modulators-wortmannin, necrosulfonamide, Z-VAD-FMK, and SBI-0206965-were applied separately. The results suggest the possible existence of bacterial programmed cell death pathways in E. coli which could be triggered by CuNP treatments.

Biological and genomic characterization of two newly isolated Elizabethkingia anophelis bacteriophages.

BACKGROUND: Elizabethkingia anophelis is an opportunistic pathogen that infects newborns and immunocompromised patients. Because the infection is associated with high mortality as a result of its intrinsic resistance to antibiotics, alternative treatment methods are needed. Our previous study successfully isolated the world's first E. anophelis phage, TCUEAP1, which showed beneficial protection to E. anophelis-infected mice. More new bacteriophages are needed in order to provide sufficient choices to combat E. anophelis infections. METHODS: In the current study, two new phages infecting E. anophelis were isolated from wastewater and were designated as TCUEAP2 and TCUEAP3. Further experiments, namely, transmission electron microscopy (TEM), infection assay, host-range analysis, and sequencing were performed to determine their biological and genomic characteristics. RESULTS: TEM analysis revealed that both TCUEAP2 and TCUEAP3 possess an icosahedral head with a non-contractile tail, and belong to the Siphoviridae family. Further experiments revealed that TCUEAP3 has a longer latent period and higher burst size compared to TCUEAP2. Host range analysis showed that both TCUEAP2 and TCUEAP3 have a narrow host range, infecting only their respective hosts. The genomic size of phage TCUEAP2 was 42,403 bps containing 61 predicted open reading frames (ORFs), whereas the genome size of TCUEAP3 was 37,073 bps containing 40 predicted ORFs. CONCLUSION: Due to the distinct biological characteristics of TCUEAP2 and TCUEAP3, they may be satisfactory for clinical uses such as preparation of phage cocktails or decontamination in clinical settings.

Visualization of the structures of the hepatitis C virus replication complex.

Hepatitis C viral RNA synthesis has been demonstrated to occur on a lipid raft membrane structure. Lipid raft membrane fraction purified by membrane flotation analysis was observed using transmission electron microscopy and atomic force microscopy. Particles around 0.7 um in size were found in lipid raft membrane fraction purified from hepatitis C virus (HCV) replicon but not their parental HuH7 cells. HCV NS5A protein was associated with these specialized particles. After several cycles of freezing-thawing, these particles would fuse into larger sizes up to 10 um. Knockdown of seven proteins associated with lipid raft (VAPA, COPG, RAB18, COMT, CDC42, DPP4, and KDELR2) of HCV replicon cells reduced the observed number of these particles and suppressed the HCV replication. Results in this study indicated that HCV replication complexes with associated lipid raft membrane form distinct particle structures of around 0.7 um as observed from transmission electron microscopy and atomic force microscopy.

Antibacterial activity of Acinetobacter baumannii phage ϕAB2 endolysin (LysAB2) against both gram-positive and gram-negative bacteria.

To investigate the nature and origin of the antibacterial activity of the lytic phage ϕAB2 toward Acinetobacter baumannii, we successfully isolated and characterized a novel phage lysozyme (endolysin) from ϕAB2 and named it LysAB2. To analyze antibacterial activity of LysAB2, the complete LysAB2 and two deletion derivatives were constructed, purified and characterized. Zymographic assays showed that only the intact LysAB2 could lyse the peptidoglycan of A. baumannii and the Staphylococcus aureus cell wall. Antibacterial analysis also showed that only the intact LysAB2 retained the complete bactericidal activity. When applied exogenously, LysAB2 exhibited a broad bacteriolytic activity against a number of Gram-negative and Gram-positive bacteria. Thermostability assays indicated that LysAB2 was stable at 20∼40 °C. Its optimal pH was 6.0, and it was active from pH 4 to 8. Scanning electron microscopy revealed that exposure to 500 µgml(-1) LysAB2 for up to 60 min caused a remarkable modification of the cell shape of the bacteria. Treating bacteria with LysAB2 clearly enhanced permeation of the bacterial cytoplasmic membrane. These results indicate that LysAB2 is an effective lysozyme against bacteria, and they suggest that it is a good candidate for a therapeutic/disinfectant agent to control nosocomial infections caused by multiple drug-resistant bacteria.

Enterovirus type 71 2A protease functions as a transcriptional activator in yeast.

Enterovirus type 71 (EV71) 2A protease exhibited strong transcriptional activity in yeast cells. The transcriptional activity of 2A protease was independent of its protease activity. EV71 2A protease retained its transcriptional activity after truncation of 40 amino acids at the N-terminus but lost this activity after truncation of 60 amino acids at the N-terminus or deletion of 20 amino acids at the C-terminus. Thus, the acidic domain at the C-terminus of this protein is essential for its transcriptional activity. Indeed, deletion of amino acids from 146 to 149 (EAME) in this acidic domain lost the transcriptional activity of EV71 2A protein though still retained its protease activity. EV71 2A protease was detected both in the cytoplasm and nucleus using confocal microscopy analysis. Coxsackie virus B3 2A protease also exhibited transcriptional activity in yeast cells. As expected, an acidic domain in the C-terminus of Coxsackie virus B3 2A protease was also identified. Truncation of this acidic domain resulted in the loss of transcriptional activity. Interestingly, this acidic region of poliovirus 2A protease is critical for viral RNA replication. The transcriptional activity of the EV71 or Coxsackie virus B3 2A protease should play a role in viral replication and/or pathogenesis.

Isolation and Characterization of a New Phage Infecting Elizabethkingia anophelis and Evaluation of Its Therapeutic Efficacy in vitro and in vivo.

Elizabethkingia spp. are a group of non-fermentative, Gram-negative, catalase-positive, and non-motile bacilli. They can cause meningitis in neonates and immunosuppressed patients, and lead to high mortality. Considering the rising trend of drug resistance among bacteria pathogens, bacteriophage (phage) therapy is a potential alternative to antibiotics for treating multidrug-resistant bacterial infections. However, so far, no phages specific for Elizabethkingia spp. have been reported. Using a clinically isolated Elizabethkingia anophelis as the host, the phage TCUEAP1 was isolated from wastewater of Hualien Tzu Chi hospital. The phage particle of TCUEAP1 under electron microscopy was revealed to belong to the siphoviridae family, with a head size of 47 nm, and a tail dimension 12 nm in diameter and 172 nm in length. The one-step growth analysis showed that the latent period of TCUEAP1 was about 40 min with a rise period lasting about 20 min, yielding a burst size of approximately 10 PFU/cell. The adsorption rate of TCUEAP1 reached about 70% in 20 min. Using 20 isolates of Elizabethkingia spp. to test the host range of TCUEAP1, it displayed narrow spectrum infecting three strains of E. anophelis, but forming spot lysis on 16 strains. The sequence result showed that the genome of TCUEAP1 is a double-stranded DNA of 49,816 bp, containing 73 predicted open reading frames. Further genomic analysis showed TCUEAP1 to be a new phage with no resemblance to publicly available phage genomes. Finally, in a mouse intraperitoneal infection model, at 6 h after the bacterial injection, TCUEAP1 decreased the bacterial load by fivefold in blood. Also, TCUEAP1 rescued 80% of mice heavily infected with E. anophelis from lethal bacteremia. We hope that the isolation and characterization of TCUEAP1, the first phage infecting Elizabethkingia spp., can promote more studies of the phages targeting this newly emerging bacterial pathogen.

Human leukocyte antigen-A, -B, and -DRB1 haplotypes of cord blood units in the Tzu Chi Taiwan Cord Blood Bank.

Cord blood (CB) is considered an alternative resource to bone marrow and peripheral blood stem cells (PBSC) for allogeneic stem cell transplantation. In this study, human leukocyte antigen (HLA)-A, -B, and -DRB1 high-resolution allele types were analyzed from a total of 710 CB units in the Tzu Chi Taiwan Cord Blood Bank. We observed 21 HLA-A alleles, 59 HLA-B alleles, and 28 HLA-DRB1 alleles, whereas 19 unique alleles were present in the CB units of 2,023 individuals selected for confirmatory testing in the Tzu Chi Taiwan Marrow Donor Registry (TCTMDR). The allelic associations between the HLA-A and -B locus were stronger than that of either the HLA-B and -DRB1 loci or the HLA-A and -DRB1 loci. The most common haplotype of CB units in the general Taiwanese population was A*3303-B*5801-DRB1*0301 (6.59%), followed by A*0207-B*4601-DRB1*0901 (3.47%) and then A*1101-B*4001-DRB1*0901 (2.11%). Moreover, two haplotypes, A*2402-B*5201-DRB1*1502 and A*0201-B*1301-DRB1*1202, existed uniquely in the CB units but were not observed in the data of TCTMDR. Although the number of CB units studied for high-resolution of HLA typing in the current study is small, we believe our data should provide useful information to increase the chances of obtaining acceptable HLA-A-, -B-, and -DRB1-matched CB units for patients.

Highly potent antimicrobial modified peptides derived from the Acinetobacter baumannii phage endolysin LysAB2.

The increase in the prevalence of multidrug-resistant Acinetobacter baumannii (MDRAB) strains is a serious public health concern. Antimicrobial peptides (AMPs) are a possible solution to this problem. In this study, we examined whether AMPs could be derived from phage endolysins. We synthesized four AMPs based on an amphipathic helical region in the C-terminus of endolysin LysAB2 encoded by the A. baumannii phage ΦAB2. These peptides showed potent antibacterial activity against A. baumannii (minimum inhibitory concentration, 4-64 µM), including some MDR and colistin-resistant A. baumannii. Of the four peptides, LysAB2 P3, with modifications that increased its net positive charge and decreased its hydrophobicity, showed high antibacterial activity against A. baumannii but little haemolytic and no cytotoxic activity against normal eukaryotic cells. The results of electron microscopy experiments and a fluorescein isothiocyanate staining assay indicated that this peptide killed A. baumannii through membrane permeabilization. Moreover, in a mouse intraperitoneal infection model, at 4 h after the bacterial injection, LysAB2 P3 decreased the bacterial load by 13-fold in ascites and 27-fold in blood. Additionally, LysAB2 P3 rescued sixty percent of mice heavily infected with A. baumannii from lethal bacteremia. Our results confirmed that bacteriophage endolysins are a promising resource for developing effective AMPs.

Rapid and sensitive detection of carbapenemase activity in Acinetobacter baumannii using superficially porous liquid chromatography-tandem mass spectrometry.

BACKGROUND: The emergence and spread of carbapenem-resistant Acinetobacter baumannii poses a challenge for optimizing antibiotic therapies and preventing outbreaks. Traditional phenotypic assays such as the modified Hodge test (MHT) or polymerase chain reaction (PCR)-based detection of the carbapenemase genes are time-consuming and complicated. Therefore, new approaches for the efficient detection of carbapenemase-producing A. baumannii are urgently required. METHODS: In this study, we used the superficially porous liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay to measure carbapenem hydrolysis in a solution spiked with test strains of A. baumannii. The rate of carbapenem hydrolysis during incubation was expressed as the ratio of the carbapenem peak area of the test A. baumannii strains to the noncarbapenemase-producing A. baumannii ATCC 17978. This method can accurately measure the carbapenem hydrolysis rate and, therefore, can effectively identify carbapenemase-producing strains within 75 minutes. RESULTS: A total of 112 A. baumannii strains were used in this study, including 103 clinical isolates with 68 carbapenem-resistant strains and 35 carbapenem-susceptible strains, seven ATCC strains and two selected mutants. The results of the superficially porous LC-MS/MS assay showed higher detection sensitivity compared to the results of the MHT. CONCLUSION: Our results demonstrate the ability of the former method to routinely detect carbapenemase-producing A. baumannii.

The Tail Associated Protein of Acinetobacter baumannii Phage ΦAB6 Is the Host Specificity Determinant Possessing Exopolysaccharide Depolymerase Activity.

Acinetobacter baumannii is a non-fermenting, gram-negative bacterium. In recent years, the frequency of A. baumannii infections has continued to increase, and multidrug-resistant strains are emerging in hospitalized patients. Therefore, as therapeutic options become limited, the potential of phages as natural antimicrobial agents to control infections is worth reconsidering. In our previous study, we isolated ten virulent double-stranded DNA A. baumannii phages, ϕAB1-9 and ϕAB11, and found that each has a narrow host range. Many reports indicate that receptor-binding protein of phage mediates host recognition; however, understanding of the specific interactions between A. baumannii and phages remains very limited. In this study, host determinants of A. baumannii phages were investigated. Sequence comparison of ϕAB6 and ϕAB1 revealed high degrees of conservation among their genes except the tail fiber protein (ORF41 in ϕAB1 and ORF40 in ϕAB6). Furthermore, we found that ORF40ϕAB6 has polysaccharide depolymerase activity capable of hydrolyzing the A. baumannii exopolysaccharide and is a component of the phage tail apparatus determining host specificity. Thus, the lytic phages and their associated depolymerase not only have potential as alternative therapeutic agents for treating A. baumannii infections but also provide useful and highly specific tools for studying host strain exopolysaccharides and producing glycoconjugate vaccines.

Identification and characterisation of the putative phage-related endolysins through full genome sequence analysis in Acinetobacter baumannii ATCC 17978.

Acinetobacter baumannii has recently emerged as a major cause of healthcare-associated infections owing to an increase in its antimicrobial resistance to virtually all available drugs. The ability of endolysins (lysozymes) to digest cell walls when applied exogenously to bacterial cells has enabled their use as novel antibacterials. In order to utilise endolysins as a therapeutic alternative to antibiotics, we surveyed the genome sequence of A. baumannii ATCC 17978 and successfully identified two phage-related endolysin genes, A1S_1600 and A1S_2016 (termed lysAB3 and lysAB4, respectively). Following cloning and expression/purification, various antibacterial activities of these two phage-related endolysins were determined in vitro. Zymographic assays showed that only purified LysAB3 could lyse the peptidoglycan of the A. baumannii cell wall. When applied exogenously, both LysAB3 and LysAB4 were active against most Acinetobacter spp. tested but had virtually no activity against other non-Acinetobacter spp. Scanning electron microscopy revealed that exposure to 100µg/mL LysAB3 and LysAB4 for up to 60min caused a remarkable modification of the cell shape of A. baumannii. These results indicate that the genes encoding phage-related endolysins can be readily isolated from the bacterial genome and have potential for the development of novel antimicrobial agents.

Isolation and characterization of phi AB2: a novel bacteriophage of Acinetobacter baumannii.

Multidrug-resistant strains of Acinetobacter baumannii (MDRAB) are increasingly being reported worldwide. Bacteriophage therapy is a potential alternative treatment for MDR bacterial infections. Although A. baumannii infection has been experimentally treated with phages, no MDRAB-specific phage has been characterized. In this study, 10 phages with differing host ranges and lysis efficacy for MDRAB were isolated; one of these, phi AB2, was further studied. Electron microscopy revealed phi AB2 to have an isometric head (60 nm), a short tail (diameter, 9 nm; length, 11 nm) and a double-stranded DNA genome--which was resistant to digestion with several restriction endonucleases--estimated to be 40 kb by pulsed-field gel electrophoresis. Partial genome sequencing of a 2.1 kb region gave sequences resembling the tubular proteins A and B of Pseudomonas aeruginosa phage LKA1. These data suggest that phi AB2 resembles phi KMV-like phages and is a new member of the Podoviridae family. It exhibited rapid adsorption (>99% adsorbed in 8 min), a short latent period (<10 min) and a large burst size (ca. 200); moreover, it was capable of infecting a wide spectrum of A. baumannii strains, causing virtually complete lysis, indicating that phi AB2 may be a good candidate as a therapeutic/disinfectant agent to control nosocomial infections caused by MDRAB.

Distributions of human leukocyte antigen-A, -B, and -DRB1 alleles and haplotypes based on 46,915 Taiwanese donors.

Human leukocyte antigen (HLA)-A, -B, and -DRB1 alleles were typed in 46,915 healthy Taiwanese volunteers recruited for Tzu Chi Taiwan Marrow Donor Registry (TCTMDR). The volunteers were separated into Taiwanese and Taiwanese aborigines. In this study, a total of 51 A, 121 B, and 53 DRB1 alleles were found in the Taiwanese group, and 17 A, 32 B, and 23 DRB1 alleles were identified in the Taiwanese aborigines. Some commonly shared alleles appeared more frequently in one group than in the other. The two haplotypes, among the 20 most frequently observed haplotypes in each group, shared in common by both groups were A*3303-B*5801-DRB1*0301 and A*0207-B*4601-DRB1* 0901. However, both haplotype frequencies in each group were extremely different, indicating the existence of genetic diversity between the two groups. In addition, principal component analysis and clustering results based on high-resolution HLA-A, -B, and -DRB1 alleles indicated that the Taiwanese group was closest to Southern Chinese and reiterated HLA diversity between the Taiwanese and the Taiwanese aborigines. We believe that our findings in this study may provide useful information in search for HLA-matched donors for patients.