Isolation and Classification of Bdellovibrio and Like Organisms

Abstract

Bdellovibrio and like organisms (BALOs) are obligate predators of Gram‐negative bacteria. BALOs are isolated as plaques growing at the expense of their prey and are cultivated as two‐member cultures. The growth cycle is composed of an extracellular attack phase and an intraperiplasmic elongation and replication phase. However, there are methods for obtaining host‐independent (HI) mutants that grow without prey on rich media. BALOs are commonly found in the environment but generally constitute small populations; therefore, their isolation may require enrichment steps. Contamination by other bacteria during isolation necessitates efficient separation between the smaller BALO cells from the majority of larger bacteria. BALOs can also be directly detected and quantified in environmental samples using specific PCR. Synchronous cultures of both wild‐type and HI derivatives can be obtained to study the different growth phases. These can be further separated by centrifugation. Classification is based on 16S rDNA analysis. Protocols relevant to these aspects of BALO detection, isolation, growth, classification, and quantitation are presented in this unit. Curr. Protoc. Microbiol. 26:7B.1.1‐7B.1.20. © 2012 by John Wiley & Sons, Inc.

Killing of anaerobic pathogens by predatory bacteria

“…BALOs used in this study (listed in Table 1) were kindly donated by Prof. E. Jurkevitch of the Hebrew University of Israel and were maintained on doublelayer HM agar plates (HEPES 25 mM, calcium 2+ 3 mM and magnesium 2+ 2 mM at pH 7.6) containing their prey organisms (Jurkevitch, 2005). BALO suspensions were serially diluted in physiological saline and 100 ll of the dilutions were mixed with 300 ll of the appropriate prey organism at a concentration of 9.70 log 10 colony-forming units (CFU) ml )1 in HM buffer.…”

Variation in genome content and predatory phenotypes betweenBdellovibriosp. NC01 isolated from soil andB. bacteriovorustype strain HD100

“…In September 2015, we collected 50 g of soil from a bioswale on the Providence College campus (lat 41.84277, lon −71.43944). To isolate predatory bacteria from the soil sample, we adapted standard methods (33, 34). We combined the soil sample with 500 ml of sterile HM buffer (25 mM HEPES adjusted to pH 7.4 and supplemented with 3 mM calcium chloride dihydrate and 2 mM magnesium chloride hexahydrate).…”

“…We obtained Bdellovibrio bacteriovorus HD100 from Mark Martin (University of Puget Sound, Puget Sound, WA) for predatory phenotype comparisons. To conduct phenotype assays, we adapted standard methods (33, 34, 53). To culture Bdellovibrio NC01 and HD100, we added a small amount of −80°C stocks to 20 ml HM buffer combined with 1.5 ml of an overnight culture of E. coli ML35.…”

Bacterial predation under changing viscosities

“…To initiate predatory co-cultures, starter predatory cultures were prepared by co-culturing the predators with their host cells (starting OD 600 = 0.2) in amended dilute nutrient broth (DNB) (a 1:10 dilution with 3 mM MgCl 2 .6H 2 O and 2 mM CaCl 2 .2H 2 O). The co-cultures were incubated at 28 C until the culture's turbidity reached below OD 600 = 0.05, corresponding to less than 10 4 CFU ml −1 of remaining prey cells and, after filtration through a 0.45 μm filter (Millipore, Billerica, MA, USA) (Jurkevitch, 2012), to 10 8 PFU ml −1 of predatory cells. Predators to be used in the experiments were obtained by inoculating 200 μl of filtered predatory cells from the starter culture (eq.…”

“…Bdellovibrio and like organisms (BALOs) are obligate predators of gram-negative bacteria. Their life cycle includes a free-living, so-called attack phase (AP) during which a small motile AP cell actively searches for a prey cell (Jurkevitch, 2012;Rotem et al, 2014). AP cells can be extremely fast, reaching velocities of up to 160 μms −1 (Lambert et al, 2006).…”