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Isolation of Bdellovibrio Sp. from Soil Samples in Mexico and Their ORIGINAL RESEARCH Isolation of Bdellovibrio sp. from soil samples in Mexico and their potential applications in control of pathogens Omotayo Opemipo Oyedara1,2, Erick de Jesus De Luna-Santillana1, Omar Olguin-Rodriguez1, Xianwu Guo1, Marco Antonio Mendoza-Villa3, Jorge Luis Menchaca-Arredondo3, Temidayo Oluyomi Elufisan1, Javier Alfonso Garza-Hernandez1, Israel Garcia Leon1 & Mario Alberto Rodriguez-Perez1 1Instituto Politécnico Nacional, Centro de Biotecnología Genómica, Reynosa, Tamaulipas 88710, México 2Department of Biological Sciences, College of Science, Engineering and Technology, Faculty of Basic and Applied Science, Osun State University, Osogbo, Osun State, Nigeria 3Facultad de Ciencias Físico-Matemáticas, Universidad Autónoma de Nuevo León, San Nicolás de los Garza, Nuevo León 66455, Mexico Keywords Abstract Atomic force microscopy, Bdellovibrio, hit locus, host-range In this study, two strains of Bdellovibrio were isolated from soil samples using the culture-dependent­ technique and two members of the family Enterobacte- Correspondence riaceae (Klebsiella sp. and Salmonella sp.) as prey. The Bdellovibrio strains were Oyedara Omotayo Opemipo, Centro de bacteriolytic, plaque-­forming, and highly motile gram-negative­ bacteria. We Biotecnología Genómica, Instituto Politécnico identified and confirmed the Bdellovibrio strains using microscopy, PCR ampli- Nacional Blvd. del Maestro Esq. Elias Piña s/n Colonia Narciso Mendoza, C.P. 88710, fication, and sequencing of the 16S rRNA gene. They were observed to be Reynosa, Tamaulipas, México. different strains based on hit locus and prey range analyses. Here, the first Tel: +52 89 9123 6245; or +234 803 report on Bdellovibrio strains isolated from soil in Mexico corroborates earlier 8438368; Fax +52 899 9243627; report indicating that populations of Bdellovibrio found in soil are heterogene- E-mail: [email protected] ous thereby the need to identify the various strains. Funding Information MA Rodriguez-Perez holds a scholarship from Comisión de Operación y Fomento de Actividades Académicas del IPN. Oyedara OO holds a doctoral scholarship from CONACYT- Mexico (Nos. 595082/326342). This work was granted by Secretaría de Investigación y Posgrado (IPN) (Project Nos. 20161059 and 20150704). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Received: 24 February 2016; Revised: 12 April 2016; Accepted: 25 April 2016 MicrobiologyOpen 2016, 5(6):992–1002 doi: 10.1002/mbo3.382 Introduction Bdellovibrio bacteriovorus, a member of the BALOs rep- resents one of the most studied predatory bacteria. It is Bdellovibrio and Like Organisms (BALOs) viz. a small (0.2–0.5 μm × 0.5–2.5 μm), uniflagellated motile ­genus Bdellovibrio, Bacteriovorax, Peredibacter, and gram-­negative bacterium that attacks and hydrolyzes cel- Halobacteriovorax are a group of obligate predatory bacteria lular constituents of other gram-­negative bacteria, utilizing that prey upon gram-­negative bacteria for nutrients and the derived nutrients for growth and reproduction. The reproduction (Snyder et al. 2002; Koval et al. 2015). life cycle of B. bacteriovorus involves attachment to a 992 © 2016 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. O. O. Oyedara et al. Soil-derived Bdellovibrio Strains from Mexico suitable gram- negative prey, loss of flagellum, followed been distributed into two different phylogenetic clades by penetration into prey periplasmic space. At this stage based on differences in features such as G+C content, of intraperiplasmic invasion, the prey cell is altered to salt tolerance and prey range (Baer et al. 2000). form a round structure known as bdelloplast. Digestion Consequently, the marine BALOs were separated from of prey cellular constituents then takes place and prior the Genus Bdellovibrio and renamed Bacteriovorax. In fact, to prey lysis, B. bacteriovorus undergoes septation to pro- the nomenclature of BALOs associated with marine habitat duce progeny that are released to carry out further preda- was recently changed from Bacteriovorax to tion (Stolp and Starr 1963; Chatterjee 2009). Halobacteriovorax (Koval et al. 2015). In the soil habitat, Several publications have indicated B. bacteriovorus as particularly, Bdellovibrio has been described to represent a better alternative to treat infections caused by multidrug- a heterogenous community of predatory bacteria that resistant bacteria (Dashiff et al. 2011; Damron and Barbier utilize wide range of gram-negative bacteria as prey 2013; Kadouri et al. 2013). It has also been suggested as (Jurkevitch et al. 2000). a biocontrol agent in aquaculture and animal husbandry Here, the first report in Mexico on molecular charac- as against the conventional antibiotics considering the terization of two Bdellovibrio strains isolated from soil increasing trend of antibiotic resistance among pathogenic using culture- dependent technique is presented. The two bacteria. Kadouri and O’Toole (2005); Monnappa et al. Bdellovibrio strains were found at a neighborhood in the (2014) reported Bdellovibrio to successfully degrade or city of Reynosa (a border city to McAllen, Texas in the inhibit biofilms produced by both gram- positive and USA), Tamaulipas State and were characterized using 16S - negative bacteria. rRNA gene sequencing, hit locus PCR amplification and B. bacteriovorus is ubiquitous in nature and may be prey range analyses. isolated from different sources including plant rhizospheres, freshwater, soil, and gastrointestinal tract of animals (Edao Materials and Methods 2000; Jurkevitch et al. 2000; Schwudke et al. 2001; Hobley et al. 2012; Lebba et al. 2013). As mentioned earlier, Sample collection B. bacteriovorus are known to be obligate intracellular predators of gram- negative bacteria hence it relies solely Soil samples were collected at two different sites on a on the degradation of prey cellular macromolecules and plot of land (26.069678N′, −98.313108W′ and utilization of derived smaller molecular materials for its 26.069446N′,−98.312902W′) within the Center for growth and reproduction. However, Bdellovibrio strains Genomic Biotechnology, National Polytechnic Institute that are capable of growing in the absence of prey as [IPN as in Spanish] located in the city of Reynosa, Mexico. well as on nutrient- rich media mostly referred to as host- The soil samples were collected with a clean hand trowel independent Bdellovibrio strains have been isolated using after removing about 20–25 mm top of soil and put into current laboratory protocols (Seidler and Starr 1969; a sterile polythene bag. The soil samples were immediately Ferguson et al. 2008). Recently, a B. bacteriovorus strain transported to the laboratory for analysis and isolation Tiberius that is capable of growing simultaneously both of Bdellovibrio strains. in the presence and absence of prey was isolated from River Tiber, Rome (Hobley et al. 2012). The molecular Determination of soil pH and electrical derivation of host-independent Bdellovibrio strains have conductivity been linked with a mutation in a genetic hit (host interaction) locus coding for proteins that play active role Soil sample was suspended in 100 mL of deionized in the attachment and invasion of Bdellovibrio into its water and stirred for 5 min. The suspension was left prey (Cotter and Thomashow 1992). In addition, about on the bench overnight (OV) and stirred again after- 89% of isolated host-independent Bdellovibrio strains have ward. It was further left for 15 min and the liquid experienced mutation in this locus, 46% demonstrated portion was transferred into a clean beaker. The pH mutation involving deletion of 42 bp on the Bd0108 which and electrical conductivity of the soil sample was then encodes protein involved in regulation of Type IV pili measured using HI 991300TM pH/EC/TDS/Temperature formation needed for Bdellovibrio attachment and invasion meters (Hanna instruments, Woonsocket, Rhode Island, (Sockett 2009; Prehna et al. 2014). USA) (Chaudhari et al. 2014). The BALOs, in general, exhibit great phylogenetic diversity and their classification have been dynamic in Host Preparation recent years owing to the distinguishing characteristics that exist between the terrestrial (including the freshwater) The bacterial preys used for preliminary isolation of and marine groups of BALOs. These two groups have the Bdellovibrio strains were Klebsiella sp. and Salmonella © 2016 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. 993 Soil-derived Bdellovibrio Strains from Mexico O. O. Oyedara et al. sp. donated by Biotechnology Institute (Autonomous earlier and concentrated at 27,000g as well as lysates were University of Nuevo Leon [UANL as in Spanish] located stored in sterile glycerol at 80°C for further study. in San Nicolás, de los Garza, Nuevo León, Mexico) and Center for Genomic Biotechnology, respectively. Microscopic identification of isolated The different preys were cultured in Luria Bertani (LB) Bdellovibrio spp using atomic force broth for 24 h and 0.4 mL was mixed with 0.1 mL microscope of filtrate for the double layer agar plating technique. For the liquid culture medium analysis, the bacterial The lysate was observed using Olympus U- TVO.35XC- 2 preys were cultured in LB broth for 24 h, harvested light
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