Label-Free Surface-Enhanced Raman Scattering Detection of Fire Blight Pathogen Using a Pathogen-Specific Bacteriophage.

Fire blight is one of the most devastating plant diseases, causing severe social and economic problems. Herein, we report a novel method based on label-free surface-enhanced Raman scattering (SERS) combined with an Erwinia amylovora-specific bacteriophage that allows detecting efficiently fire blight bacteria E. amylovora for the first time. To achieve the highest SERS signals for E. amylovora, we synthesized and compared plasmonic nanoparticles (PNPs) with different sizes, i.e., bimetallic gold core-silver shell nanoparticles (Au@AgNPs) and monometallic gold nanoparticles (AuNPs) and utilized the coffee-ring effect for the self-assembly of PNPs and enrichment of fire blight bacteria. Furthermore, we investigated the changes in the SERS spectra of E. amylovora after incubation with an E. amylovora-specific bacteriophage, and we found considerable differences in the SERS signals as a function of the bacteriophage incubation time. The results indicate that our bacteriophage-based label-free SERS analysis can specifically detect E. amylovora without the need for peak assignment on the SERS spectra but simply by monitoring the changes in the SERS signals over time. Therefore, our facile method holds great potential for the label-free detection of pathogenic bacteria and the investigation of viral-bacterial interactions.

Bacteriophage cocktail for biocontrol of soft rot disease caused by Pectobacterium species in Chinese cabbage.

Pectobacterium spp. are necrotrophic plant pathogens that cause the soft rot disease in Chinese cabbage, resulting in severe yield loss. The use of conventional antimicrobial agents, copper-based bactericides, and antibiotics has encountered several limitations, such as bioaccumulation on plants and microbial resistance. Bacteriophages (phages) are considered promising alternative antimicrobial agents against diverse phytopathogens. In this study, we isolated and characterized two virulent phages (phiPccP-2 and phiPccP-3) to develop a phage cocktail. Morphological and genomic analyses revealed that two phages belonged to the Tevenvirinae and Mccorquodalevirinae subfamilies, respectively. The phiPccP-2 and phiPccP-3 phages, which have a broad host range, were stable at various environmental conditions, such as various pHs and temperatures and exposure to ultraviolet light. The phage cocktail developed using these two lytic phages inhibited the emergence of phage-resistant bacteria compared to single-phage treatments in in vitro challenge assays. The phage cocktail treatment effectively prevented the development of soft rot symptom in matured Chinese cabbage leaves. Additionally, the phage cocktail comprising three phages (phiPccP-1, phiPccP-2, and phiPccP-3) showed superior biocontrol efficacy against the mixture of Pectobacterium strains in Chinese cabbage seedlings. These results suggest that developing phage cocktails is an effective approach for biocontrol of soft rot disease caused by Pectobacterium strains in crops compared to single-phage treatments. KEY POINTS: •Two newly isolated Pectobacterium phages, phiPccP-2 and phiPccP-3, infected diverse Pectobacterium species and effectively inhibited the emergence of phage-resistant bacteria. •Genomic and physiological analyses suggested that both phiPccP-2 and phiPccP-3 are lytic phages and that their lytic activities are stable in the environmental conditions under which Chinese cabbage grows. •Treatment using a phage cocktail comprising phiPccP-2 and phiPccP-3 efficiently suppressed soft rot disease in detached mature leaves and seedlings of Chinese cabbage, indicating the applicability of the phage cocktail as an alternative antimicrobial agent.

On-site applicable diagnostic fluorescent probe for fire blight bacteria.

Fire blight is a representative plant infection that contaminates edible plants and causes socio-economic problems in agricultural and livestock industries globally. It is caused by the pathogen Erwinia amylovora (E. amylovora) creates lethal plant necrosis and spreads rapidly across plant organs. We newly disclose the fluorogenic probe B-1 for real-time on-site detection of fire blight bacteria for the first time. B-1 exhibited no emission signals but manifested bright emission properties in the presence of fire blight bacteria. Based on these features, fluorescence imaging of the fire blight bacteria and its real-time detection from the infected host plant tissues were conducted. The detection limit against E. amylovora was 102 CFU/mL, which had excellent sensitivity. The fluorogenic probe-based on-site diagnostic technology was supplemented by introducing a new portable UV device. This work holds enormous potential to be a new advanced tool for detecting fire blight in agricultural and livestock industries.

Phage PPPL-1, A New Biological Agent to Control Bacterial Canker Caused by Pseudomonas syringae pv. actinidiae in Kiwifruit.

Pseudomonas syringae pv. actinidiae (Psa) is a Gram-negative bacterium that causes bacterial canker disease in kiwifruit. Copper or antibiotics have been used in orchards to control this disease, but the recent emergence of antibiotic-resistant Psa has called for the development of a new control agent. We previously reported that the bacteriophage (or phage) PPPL-1 showed antibacterial activity for both biovar 2 and 3 of Psa. To investigate the possibility of PPPL-1 to control bacterial canker in kiwifruit, we further tested the efficacy of PPPL-1 and its phage cocktail with two other phages on suppressing disease development under greenhouse conditions using 6 weeks old kiwifruit plants. Our results showed that the disease control efficacy of PPPL-1 treatment was statistically similar to those of phage cocktail treatment or AgrimycinTM, which contains streptomycin and oxytetracycline antibiotics as active ingredients. Moreover, PPPL-1 could successfully kill streptomycin-resistant Psa isolates, of which the treatment of BuramycinTM carrying only streptomycin as an active ingredient had no effect in vitro. The phage PPPL-1 was further characterized, and stability assays showed that the phage was stable in the field soil and at low temperature of 0 ± 2 °C. In addition, the phage could be scaled up quickly up to 1010 pfu/mL at 12 h later from initial multiplicity of infection of 0.000005. Our results indicate that PPPL-1 phage is a useful candidate as a biocontrol agent and could be a tool to control the bacterial canker in kiwifruit by Psa infection in the field conditions.

Biocontrol of Soft Rot Caused by Pectobacterium odoriferum with Bacteriophage phiPccP-1 in Kimchi Cabbage.

Pectobacterium odoriferum has recently emerged as a widely infective and destructive pathogen causing soft-rot disease in various vegetables. Bacteriophage phiPccP-1 isolated from Pyeongchang, South Korea, showed lytic activity against P. odoriferum Pco14 and two other Pectobacterium species. The transmission electron microscopy and genome phylograms revealed that phiPccP-1 belongs to the Unyawovirus genus, Studiervirinae subfamily of the Autographivirinae family. Genome comparison showed that its 40,487 bp double-stranded DNA genome shares significant similarity with Pectobacterium phage DU_PP_II with the identity reaching 98% of the genome. The phiPccP-1 application significantly inhibited the development of soft-rot disease in the mature leaves of the harvested Kimchi cabbage up to 48 h after Pco14 inoculation compared to the untreated leaves, suggesting that phiPccP-1 can protect Kimchi cabbage from soft-rot disease after harvest. Remarkably, bioassays with phiPccP-1 in Kimchi cabbage seedlings grown in the growth chamber successfully demonstrated its prophylactic and therapeutic potential in the control of bacterial soft-rot disease in Kimchi cabbage. These results indicate that bacteriophage phiPccP-1 can be used as a potential biological agent for controlling soft rot disease in Kimchi cabbage.

Bacteriophage Usage for Bacterial Disease Management and Diagnosis in Plants.

In nature, plants are always under the threat of pests and diseases. Pathogenic bacteria are one of the major pathogen types to cause diseases in diverse plants, resulting in negative effects on plant growth and crop yield. Chemical bactericides and antibiotics have been used as major approaches for controlling bacterial plant diseases in the field or greenhouse. However, the appearance of resistant bacteria to common antibiotics and bactericides as well as their potential negative effects on environment and human health demands bacteriologists to develop alternative control agents. Bacteriophages, the viruses that can infect and kill only target bacteria very specifically, have been demonstrated as potential agents, which may have no negative effects on environment and human health. Many bacteriophages have been isolated against diverse plant-pathogenic bacteria, and many studies have shown to efficiently manage the disease development in both controlled and open conditions such as greenhouse and field. Moreover, the specificity of bacteriophages to certain bacterial species has been applied to develop detection tools for the diagnosis of plant-pathogenic bacteria. In this paper, we summarize the promising results from greenhouse or field experiments with bacteriophages to manage diseases caused by plant-pathogenic bacteria. In addition, we summarize the usage of bacteriophages for the specific detection of plant-pathogenic bacteria.

Population structure of colonizing and invasive Staphylococcus aureus strains in northern Vietnam.

Staphylococcus aureus is an important global health problem worldwide. There is still scarce information on the population structure of S. aureus strains in Asia, where the majority of the world population lives. This study characterized the diversity of S. aureus strains in northern Vietnam through multilocus sequence typing (MLST). Eighty-five carriage isolates from the community and 77 invasive isolates from the clinical setting were selected and tested for meticillin resistance and the presence of Panton-Valentine leukocidin (PVL). MLST was performed on these isolates, of which CC59 (25.4 %), CC188 (17.3 %) and CC45 (16.7 %) were the predominant clonal complexes (CCs). CC59 carriage isolates had significantly lower rates of meticillin-resistant S. aureus (MRSA) than their corresponding clinical group isolates (32 vs 83 %). There were no significant differences in rates of MRSA between carriage isolates and clinical isolates of CC45 and CC188. CC59 carriage isolates were significantly lower in rates of PVL+ than CC59 clinical isolates (32 vs 83 %), but the converse was shown in CC45 isolates (14 vs 0 %, respectively). This study revealed vast differences in the molecular epidemiology and population structure of S. aureus in community and clinical settings in Vietnam. Nevertheless, the data underline the spread of virulent and/or resistant strains (MRSA and/or PVL+) in the community, suggesting the necessity for further surveillance to determine the mechanism of transmission of these strains (i.e. MRSA/PVL+) outside clinical settings.

Field tested milliliter-scale blood filtration device for point-of-care applications.

In this paper, we present a low cost and equipment-free blood filtration device capable of producing plasma from blood samples with mL-scale capacity and demonstrate its clinical application for hepatitis B diagnosis. We report the results of in-field testing of the device with 0.8-1 ml of undiluted, anticoagulated human whole blood samples from patients at the National Hospital for Tropical Diseases in Hanoi, Vietnam. Blood cell counts demonstrate that the device is capable of filtering out 99.9% of red and 96.9% of white blood cells, and the plasma collected from the device contains lower red blood cell counts than plasma obtained from a centrifuge. Biochemistry and immunology testing establish the suitability of the device as a sample preparation unit for testing alanine transaminase (ALT), aspartate transaminase (AST), urea, hepatitis B "e" antigen (HBeAg), hepatitis B "e" antibody (HBe Ab), and hepatitis B surface antibody (HBs Ab). The device provides a simple and practical front-end sample processing method for point-of-care microfluidic diagnostics, enabling sufficient volumes for multiplexed downstream tests.

Hand-powered microfluidics: A membrane pump with a patient-to-chip syringe interface.

In this paper, we present an on-chip hand-powered membrane pump using a robust patient-to-chip syringe interface. This approach enables safe sample collection, sample containment, integrated sharps disposal, high sample volume capacity, and controlled downstream flow with no electrical power requirements. Sample is manually injected into the device via a syringe and needle. The membrane pump inflates upon injection and subsequently deflates, delivering fluid to downstream components in a controlled manner. The device is fabricated from poly(methyl methacrylate) (PMMA) and silicone, using CO2 laser micromachining, with a total material cost of ∼0.20 USD/device. We experimentally demonstrate pump performance for both deionized (DI) water and undiluted, anticoagulated mouse whole blood, and characterize the behavior with reference to a resistor-capacitor electrical circuit analogy. Downstream output of the membrane pump is regulated, and scaled, by connecting multiple pumps in parallel. In contrast to existing on-chip pumping mechanisms that typically have low volume capacity (∼5 µL) and sample volume throughput (∼1-10 µl/min), the membrane pump offers high volume capacity (up to 240 µl) and sample volume throughput (up to 125 µl/min).

Etiology and epidemiology of diarrhea in children in Hanoi, Vietnam.

OBJECTIVES: This paper provides a preliminary picture of diarrhea with regards to etiology, clinical symptoms, and some related epidemiologic factors in children less than five years of age living in Hanoi, Vietnam. METHODS: The study population included 587 children with diarrhea and 249 age-matched healthy controls. The identification of pathogens was carried out by the conventional methods in combination with ELISA, immunoseparation, and PCR. The antibiotic susceptibility was determined by MIC following the NCCLS recommendations. RESULTS: Of those with diarrhea, 40.9% were less than one year old and 71.0% were less than two years old. A potential pathogen was identified in 67.3% of children with diarrhea. They were group A rotavirus, diarrheagenic Escherichia coli, Shigella spp, and enterotoxigenic Bacteroides fragilis, with prevalences of 46.7%, 22.5%, 4.7%, and 7.3%, respectively. No Salmonella spp or Vibrio cholerae were isolated. Rotavirus and diarrheagenic E. coli were predominant in children less than two years of age, while Shigella spp, and enterotoxigenic B. fragilis were mostly seen in the older children. Diarrheagenic E. coli and Shigella spp showed high prevalence of resistance to ampicillin, chloramphenicol, and to trimethoprim/sulfamethoxazole. Children attending the hospitals had fever (43.6%), vomiting (53.8%), and dehydration (82.6%). Watery stool was predominant with a prevalence of 66.4%, followed by mucous stool (21.0%). The mean episodes of stools per day was seven, ranging from two to 23 episodes. Before attending hospitals, 162/587 (27.6%) children had been given antibiotics. Overall, more children got diarrhea in (i) poor families; (ii) families where piped water and a latrine were lacking; (iii) families where mothers washed their hands less often before feeding the children; (iv) families where mothers had a low level of education; (v) families where information on health and sanitation less often reached their households. CONCLUSIONS: Group A rotavirus, diarrheagenic Escherichia coli, Shigella spp, and enterotoxigenic Bacteroides fragilis play an important role in causing diarrhea in children in Hanoi, Vietnam. Epidemiological factors such as lack of fresh water supply, unhygienic septic tank, low family income, lack of health information, and low educational level of parents could contribute to the morbidity of diarrhea in children.

Diarrhea caused by enterotoxigenic Bacteroides fragilis in children less than 5 years of age in Hanoi, Vietnam.

Enterotoxigenic Bacteroides fragilis (ETBF) are considered as an emerging enteropathogen causing diarrhea in children. Eight hundred and thirty-six (836) children less than 5 years of age including 587 children with diarrhea and 249 age-matched controls were involved in the study. Within the group of children with diarrhea, 7.3% (43/587) ETBF was detected by immunoseparation in combination with polymerase chain reaction. The corresponding figure for the controls was 2.4% (6/249) (P<0.01). Within the diarrhea group, the prevalence was significantly higher in children older than 1 year of age. Three subtypes of ETBF isolates have been identified with the prevalence of 67.4%, 18.6%, and 16% for bft-1, bft-2, and a new bft, respectively. In the controls, two of the subtypes were identified, 5 bft-1 and 1 bft-2. More than half (55.8%) of the samples harboring ETBF also had other identified pathogens. The clinical symptoms of the single ETBF infection were not different from those of co-infections. This is the first study of the role of ETBF in children's diarrhea in Vietnam and it is concluded that this pathogen is an important causative agent of diarrhea in children in Hanoi, Vietnam.