Phenotypic characterization of phage vB_vcM_Kuja.

Bacteriophage therapy targeting the increasingly resistant Vibrio cholerae is highly needed. Hence, studying the phenotypic behavior of potential phages under different conditions is a prerequisite to delivering the phage in an active infective form. The objective of this study was to characterize phage VP4 (vB_vcM_Kuja), an environmental vibriophage isolated from River Kuja in Migori County, Kenya in 2015. The phenotypic characteristics of the phage were determined using a one-step growth curve, restriction digestion profile, pH, and temperature stability tests. The results revealed that the phage is stable through a wide range of temperatures (20-50°C) and maintains its plaque-forming ability at pH ranging from 6 to 12. The one-step growth curve showed a latent period falling between 40 and 60 min, while burst size ranged from 23 to 30 plaque-forming units/10 µl at the same host strain. The restriction digestion pattern using EcoRI, SalI, HindIII, and XhoI enzymes showed that HindIII could cut the phage genome. The phage DNA could not be restricted by the other three enzymes. The findings of this study can be used in future studies to determine phage-host interactions.

Novel PhoH-encoding vibriophages with lytic activity against environmental Vibrio strains.

Cholera is a devastating diarrheal disease that accounts for more than 10% of children's lives worldwide, but its treatment is hampered by a rise in antibiotic resistance. One promising alternative to antibiotic therapy is the use of bacteriophages to treat antibiotic-resistant cholera infections, and control Vibrio cholera in clinical cases and in the environment, respectively. Here, we report four novel, closely related environmental myoviruses, VP4, VP6, VP18, and VP24, which we isolated from two environmental toxigenic Vibrio cholerae strains from river Kuja and Usenge beach in Kenya. High-throughput sequencing followed by bioinformatics analysis indicated that the genomes of the four bacteriophages have closely related sequences, with sizes of 148,180 bp, 148,181 bp, 148,179 bp, and 148,179 bp, and a G + C content of 36.4%. The four genomes carry the phoH gene, which is overrepresented in marine cyanophages. The isolated phages displayed a lytic activity against 15 environmental, as well as one clinical, Vibrio cholerae strains. Thus, these novel lytic vibriophages represent potential biocontrol candidates for water decontamination against pathogenic Vibrio cholerae and ought to be considered for future studies of phage therapy.

Bacteriophages Isolated in China for the Control of Pectobacterium carotovorum Causing Potato Soft Rot in Kenya.

Soft rot is an economically significant disease in potato and one of the major threats to sustainable potato production. This study aimed at isolating lytic bacteriophages and evaluating methods for and the efficacy of applying phages to control potato soft rot caused by Pectobacterium carotovorum. Eleven bacteriophages isolated from soil and water samples collected in Wuhan, China, were used to infect P. carotovorum host strains isolated from potato tubers showing soft rot symptoms in Nakuru county, Kenya. The efficacy of the phages in controlling soft rot disease was evaluated by applying individual phage strains or a phage cocktail on potato slices and tubers at different time points before or after inoculation with a P. carotovorum strain. The phages could lyse 20 strains of P. carotovorum, but not Pseudomonas fluorescens control strains. Among the 11 phages, Pectobacterium phage Wc5r, interestingly showed cross-activity against Pectobacterium atrosepticum and two phage-resistant P. carotovorum strains. Potato slice assays showed that the phage concentration and timing of application are crucial factors for effective soft rot control. Phage cocktail applied at a concentration of 1 × 109 plaque-forming units per milliliter before or within an hour after bacterial inoculation on potato slices, resulted in ≥ 90% reduction of soft rot symptoms. This study provides a basis for the development and application of phages to reduce the impact of potato soft rot disease.

Developing a bacteriophage cocktail for biocontrol of potato bacterial wilt.

Bacterial wilt is a devastating disease of potato and can cause an 80% production loss. To control wilt using bacteriophage therapy, we isolated and characterized twelve lytic bacteriophages from different water sources in Kenya and China. Based on the lytic curves of the phages with the pathogen Ralstonia solanacearum, one optimal bacteriophage cocktail, P1, containing six phage isolations was formulated and used for studying wilt prevention and treatment efficiency in potato plants growing in pots. The preliminary tests showed that the phage cocktail was very effective in preventing potato bacterial wilt by injection of the phages into the plants or decontamination of sterilized soil spiked with R. solanacearum. Eighty percent of potato plants could be protected from the bacterial wilt (caused by R. solanacearum reference strain GIM1.74 and field isolates), and the P1 cocktail could kill 98% of live bacteria spiked in the sterilized soil at one week after spraying. However, the treatment efficiencies of P1 depended on the timing of application of the phages, the susceptibility of the plants to the bacterial wilt, as well as the virulence of the bacteria infected, suggesting that it is important to apply the phage therapy as soon as possible once there are early signs of the bacterial wilt. These results provide the basis for the development of bacteriophagebased biocontrol of potato bacterial wilt as an alternative to the use of antibiotics.

Morphological and Genetic Diversity of Rhizobia Nodulating Cowpea (Vigna unguiculata L.) from Agricultural Soils of Lower Eastern Kenya.

Limited nitrogen (N) content in the soil is a major challenge to sustainable and high crop production in many developing countries. The nitrogen fixing symbiosis of legumes with rhizobia plays an important role in supplying sufficient N for legumes and subsequent nonleguminous crops. To identify rhizobia strains which are suitable for bioinoculant production, characterization of rhizobia is a prerequisite. The objective of this study was to assess the morphological and genetic diversity of rhizobia that nodulates cowpea in agricultural soils of lower eastern Kenya. Twenty-eight rhizobia isolates were recovered from soil samples collected from farmers' fields in Machakos, Makueni, and Kitui counties in lower eastern Kenya and characterized based on morphological characteristics. Thirteen representative isolates were selected and characterized using BOX repetitive element PCR fingerprinting. Based on the dendrogram generated from morphological characteristics, the test isolates were distributed into two major clusters at a similarity of 75%. Phylogenetic tree, based on BOX repetitive element PCR, grouped the isolates into two clusters at 90% similarity level. The clustering of the isolates did not show a relationship to the origin of soil samples, although the isolates were genetically diverse. This study is a prerequisite to the selection of suitable cowpea rhizobia to develop bioinoculants for sustainable crop production in Kenya.

Characterization of Vibrio cholerae bacteriophages isolated from the environmental waters of the Lake Victoria region of Kenya.

Over the last decade, cholera outbreaks have become common in some parts of Kenya. The most recent cholera outbreak occurred in Coastal and Lake Victoria region during January 2009 and May 2010, where a total of 11,769 cases and 274 deaths were reported by the Ministry of Public Health and Sanitation. The objective of this study is to isolate Vibrio cholerae bacteriophages from the environmental waters of the Lake Victoria region of Kenya with potential for use as a biocontrol for cholera outbreaks. Water samples from wells, ponds, sewage effluent, boreholes, rivers, and lakes of the Lake Victoria region of Kenya were enriched for 48 h at 37 °C in broth containing a an environmental strain of V. cholerae. Bacteriophages were isolated from 5 out of the 42 environmental water samples taken. Isolated phages produced tiny, round, and clear plaques suggesting that these phages were lytic to V. cholerae. Transmission electron microscope examination revealed that all the nine phages belonged to the family Myoviridae, with typical icosahedral heads, long contractile tails, and fibers. Head had an average diameter of 88.3 nm and tail of length and width 84.9 and 16.1 nm, respectively. Vibriophages isolated from the Lake Victoria region of Kenya have been characterized and the isolated phages may have a potential to be used as antibacterial agents to control pathogenic V. cholerae bacteria in water reservoirs.