Abstract
The present study investigated the predatory efficacy of Bdellovibrio isolated from untreated hospital wastewater sources against human pathogens. Bdellovibrio is a Gram-negative, motile, uniflagellate predatory bacteria present in the environment, which directly predates on other bacteria, including human pathogens. In the present study, 30 hospital effluent samples were collected and screened for Bdellovibrio. A total of 11 Bdellovibrio isolates were obtained by the double-layer agar technique. All the isolates were identified by amplification of the 16S rDNA coding region using polymerase chain reaction (PCR) and confirmed as Bdellovibrio stolpii. The lytic activity of confirmed isolates was investigated against four Gram-negative bacteria Pseudomonas aeruginosa ADW44, Pseudomonas aeruginosa 27853(ATCC Strain), V. cholerae and Salmonella typhimurium of clinical origin obtained from the institutional repository. Among the 11 isolates, three Bdellovibrio isolates NBd1, YBd1 and RBd1 demonstrated the ability to prey upon the tested clinical isolates. To the best of our knowledge, this is the first report on the isolation of B. stolpii from hospital wastewater settings in India with broad and high bacteriolytic activity.
HIGHLIGHT
Antibiotic resistance in bacteria is a major concern in both national and international scenarios.
The study gives insights into the predatory spectrum of Bdellovibrio, against multidrug-resistant bacteria.
The present study may help in the development of Bdellovibrio as a whole-organism approach against biofilm-forming bacteria.
Developing more efficient, economic, and environmentally friendly biocontrol agents against multidrug-resistant bacteria is the need of the hour.
Graphical Abstract
INTRODUCTION
Growing concerns among a greater number of individuals about the emergence of drug resistance within bacterial communities are being raised due to the extensive use and abuse of antimicrobial agents. In addition to drug resistance, bacterial biofilms being resistant to antimicrobial agents is another threat. The treatment of infections caused by the biofilm formers is a major challenge since these bacteria are protected well inside the biofilm which results in more resistance to antibiotic treatment. Extensive research has revealed its complex mechanism of infection and treatment strategies are limited because of a lack of sensitive detection methods and a narrow range of effective antibiotics. Facing the limitations of antibiotics, there is an increasing need for the development of other alternative methods for the efficient control of clinical pathogens and managing bacterial infections caused by these pathogens. Bdellovibrio is a predatory bacterium present in the environment that directly predates on other bacteria which overviews the concept of a biocontrol agent.
Bdellovibrio is a group of obligatory predatory bacteria which prey upon other bacteria especially Gram-negative bacteria for nutrients and reproduction. The organism was first discovered by Stolp & Starr (1963) from the soil during the isolation of Pseudomonas bacteriophage. Bdellovibrio is a small Gram-negative bacteria measuring 0.2–0.5 μm × 0.5–2.5 μm in size, highly motile with single-sheathed polar flagella (Kadouri & O'Toole 2005). Bdellovibrio predates on other larger Gram-negative bacteria by invading their periplasmic space, degrading the prey cellular macromolecules and undergoing a complex replication cycle releasing the progeny (Rendulic et al. 2004; Caulton & Lovering 2020). They exhibit a dimorphic cell cycle with several events such as prey location, attachment, penetration, establishment, elongation, development (septa formation) and progeny release (Lambert et al. 2006). The lytic action of the Bdellovibrio species rapidly reduces prey populations.
Bdellovibrio species are ubiquitous in nature and are found in different environmental sources such as soil, freshwater, rhizosphere and the gastrointestinal tract of animals (Sar et al. 2015; Oyedara et al. 2016). The strains also have the capacity to grow in the absence of prey as well as in the presence of nutrient-rich media using laboratory protocols (Ferguson et al. 2008). The study provided an important insight into the significance of predatory bacteria in the shaping of community structure, its impact on complex communities and its function in both natural and engineered ecosystems (Feng et al. 2016). The predation efficiency of Bdellovibrio was evaluated against six different periodontal pathogens (Van Essche et al. 2011). The strain Bdellovibrio (HD100) markedly reduced the biofilm formation and was used to combat periodontal pathogens (Van Essche et al. 2009).
Bdellovibrio with several prospective applications as biocontrol agents in aquaculture, animal husbandry and medicine is gaining momentum considering the increasing trend of antibiotic resistance among pathogenic bacteria and the use of living predatory bacteria is one of the potential approaches to overcome this global health and economic crisis (Atterbury & Tyson 2021). Even though interest in predatory bacteria has recently surged, only Bdellovibrio bacteriovorus species is studied best among BALOs (Bdellovibrio-like organisms), and serves as a model organism for bacterial predation (Bratanis et al. 2020). Many of the molecular mechanisms during prey invasion, mode of nutrient acquisition, and details on their importance of predation remain partial and rather elusive. The emergence of newer multidrug-resistant (MDR) pathogens pays a severe burden on the health infrastructure of developing countries. There is a dearth of local findings on Bdellovibrio in India and their predatory activities against clinical isolates to use them as a whole organism approach.
Bdellovibrio strains exhibit obligatory lifestyle and selectively prey on a broad range of Gram-negative bacteria, including MDR pathogens. The kinetic lysis of the Bdellovibrio as a predator to antibiotic-resistant Salmonella paratyphi as prey organisms was demonstrated and suggested their use of biocontrol agents in water (El-Shanshoury et al. 2016). Most of the published research used pure cultures of Bdellovibrio bacteriovorus to elucidate the prey–predator interaction. The predatory behavior of these Bdellovibrio in complex natural habitats is still to be studied. Therefore, this study would significantly contribute to the existing database of information on Bdellovibrio and thereby may help in bringing a step closer to the eventual widespread applications of Bdellovibrio stolpii for the treatment of infections in man. The isolation of Bdellovibrio from different environmental sources like soil and water was reported in previous studies (Rogosky et al. 2006; Markelova & Kerzhentsev 1998; Chu & Zhu 2010). Predatory activities of isolated Bdellovibrio were evaluated against periodontal pathogens and aquaculture isolates (Van Essche et al. 2011). Bdellovibrio has more advantages over bacteriophages with multiple preys and the capability to invade biofilms. Dual predation by bacteriophage and Bdellovibrio in the eradication of Escherichia coli prey was more successful where individual predation cannot (Hobley et al. 2020). Detection of Bdellovibrio in hospital wastewater and evaluation of predatory efficacy against the clinical isolates has not been elaborately elucidated before. The present study would therefore give insights into the predatory spectrum of isolated Bdellovibrio which may help in the development of Bdellovibrio as a whole organism approach in treating the infections caused by biofilm-forming clinical pathogens. Hence, the present study was undertaken to isolate Bdellovibrio from untreated wastewater samples and to evaluate the inhibitory activity against selected Gram-negative clinical pathogens obtained from the institutional repository.
MATERIALS AND METHODS
Sample collection
Sterile 100 ml containers with stoppers were used for sample collection. Untreated wastewater samples were collected from two tertiary care hospitals located in and around Mangaluru (from the outlet pipe before the treatment). A total of 30 effluents were collected during the study period from December 2020 to June 2021 and processed for Bdellovibrio isolation within 4 h after sample collection.
Prey cell preparation for Bdellovibrio isolation
Isolation of Bdellovibrio
The untreated hospital wastewater samples collected were centrifuged at 6,000 rpm for 5 min at 4 °C to remove suspended materials. The resulting supernatant was filtered through a 0.45 μm syringe filter to remove all the larger bacteria. The filtrate was again centrifuged at 12,000 rpm for 20 min at 4 °C to pellet out the Bdellovibrio. The supernatant was discarded and the pellets were resuspended in 1 ml of HEPES buffer. 1 ml of this suspension was used for the double-layer agar technique wherein it was mixed with 4 ml of dilute nutrient broth agar (0.7% agar) and 0.2 ml of prey culture and plated on dilute nutrient agar (1.5% agar). The plates were incubated at 30 °C and observed for 3–7 days for the development of the growing plaques (Oyedara et al. 2016). Potential Bdellovibrio plaques were defined as those that appear between 48 and 72 h and are observed for increased zone size.
Single plaque isolation
The plaques which appeared between 48 and 72 h were transferred to 5 ml of dilute nutrient broth containing prey cells. This culture was incubated at 30 °C for 48 h to monitor the clearance of the suspension. The clear culture obtained was centrifuged at 8,000 rpm at 4 °C for 5 min to remove prey cells and the supernatant was further subjected to centrifugation at 12,000 rpm for 30 min at 4 °C. This step was repeated twice and the pure culture of Bdellovibrio was preserved at −80 °C in 40% glycerol broth (Oyedara et al. 2016).
Molecular identification of Bdellovibrio isolates
For molecular identification, amplification of 16S rDNA gene and hit locus gene was done using polymerase chain reaction (PCR). DNA was extracted from each Bdellovibrio isolate by CTAB (Cetyl trimethyl ammonium bromide) method. Briefly, Bdellovibrio isolates were inoculated into 5 ml of dilute nutrient broth with prey cells and incubated at 30 °C for 48 h. The prey cells were removed by centrifugation at 8,000 rpm for 5 min and further centrifuged at 12,000 rpm for 30 min to pellet out Bdellovibrio cells. To this pellet, 400 μl of TE buffer and 100 μl of NaCl (5 M) were added and mixed well by vortexing. To this, 50 μl of CTAB was added and incubated at 60 °C for 20 min. After the incubation, 500 μl of chloroform was added, mixed well and incubated at 4 °C for 30 min. The incubated suspension was centrifuged at 12,000 rpm for 10 min and the resulting supernatant was collected into a new centrifuge tube. To the supernatant collected, 500 μl of phenol:chloroform:isoamyl alcohol (25:24:1) was added, mixed well and centrifuged at 12,000 rpm for 5 min. The supernatant thus obtained was transferred to a new centrifuge tube and 500 μl of chloroform was added, vortexed and centrifuged at 12,000 rpm for 5 min. To the supernatant obtained, in a fresh centrifuge tube, 1/10 volume of Na-acetate (50 μl) and 2/10 volume of ice-cold ethanol (1,000 μl) were added to precipitate DNA and incubated in ice for 15 min and centrifuged at 12,000 rpm for 10 min. To the pellet, 500 μl of 70% ethanol was added and centrifuged at 10,000 rpm for 5 min. The supernatant was discarded and tubes were inverted on a paper towel to remove traces of ethanol. The resulting pellet was suspended into 50 μl of elution buffer and stored as final DNA preparation at −20 °C and was used as a template in PCR reaction.
A volume of 30 μl of the reaction mixture was used for PCR to check the presence of 16S rDNA for genus-level identification and further species-level identification was performed using specific primers described by Varon & Shilo (1981). All the PCR reactions were carried out in a PCR system (Bio-Rad, CA, USA, Model: S1000). The details of the primers are listed in Table 1.
PCR primer details used in this study for molecular identification of Bdellovibrio
Primers . | Sequence . | Annealing Temp (°C) . | Product size (in bp) . |
---|---|---|---|
Bd | F: AGAGTTTGATTCTGGCTCAGA R: AGGTGATCCAGCCGCAGGTTC | 62 | ∼1,495 |
Hit | F: GTGGCTTCAAACGGAGTGGA R: ACGACTGTGAACGGCAACG | 56 | ∼700 |
BdBac | F: GCGTGCCTAATACATGCAAG R: AGATAGCTTTTAAGCGATTTGCTCTA | 45 | ∼1,200 |
BdSp | F: GCGTGCCTAATACATGCAAG R: CGGTTTTTTGAGATTGGCTC | 45 | ∼1,200 |
BdSr | F: GCGTGCCTAATACATGCAAG R: CCGAACTGAGGCGCGC | 45 | ∼1,200 |
Primers . | Sequence . | Annealing Temp (°C) . | Product size (in bp) . |
---|---|---|---|
Bd | F: AGAGTTTGATTCTGGCTCAGA R: AGGTGATCCAGCCGCAGGTTC | 62 | ∼1,495 |
Hit | F: GTGGCTTCAAACGGAGTGGA R: ACGACTGTGAACGGCAACG | 56 | ∼700 |
BdBac | F: GCGTGCCTAATACATGCAAG R: AGATAGCTTTTAAGCGATTTGCTCTA | 45 | ∼1,200 |
BdSp | F: GCGTGCCTAATACATGCAAG R: CGGTTTTTTGAGATTGGCTC | 45 | ∼1,200 |
BdSr | F: GCGTGCCTAATACATGCAAG R: CCGAACTGAGGCGCGC | 45 | ∼1,200 |
Specific amplification was performed using a thermocycler with the following PCR conditions: initial denaturation at 95 °C for 5 min, 30 cycles of denaturation at 95 °C for 30 s, annealing at 62 °C (Bd primer) 45 °C (BdBac, BdSp and BdSr primers) and 54 °C (Hit primer) for 30 s, extension at 72 °C for 45 s, and final extension at 72 °C for 10 min. The PCR products after amplification were subjected to gel electrophoresis. Briefly, 2% agarose gel, stained with ethidium bromide (0.5 μg/ml) was visualized under ultraviolet light using a gel documentation system (Bio-Rad, CA, USA, Model: Gel DocTMXR+ ). All positive PCR products were purified and sent for sequencing at Eurofins Genomics India Pvt Ltd, Bangalore. The obtained sequences were compared to gene sequences in GenBank by means of the Basic Local Alignment Search Tool (BLAST) present at the National Centre for Biotechnology Information (NCBI) website (http:// www.ncbi.nlm.nih.gov).
Antibiotic resistance profile of Gram-negative pathogens
Antibiotic susceptibility for the test organisms Salmonella typhimurium, Vibrio cholerae, P. aeruginosa ADW44 and P. aeruginosa 27853 (ATCC strain) was performed using the method described by Bauer et al. (1966) on Muller–Hinton agar as recommended by Clinical and Laboratory Standards Institute (CLSI 2017) guidelines. The antibiotics used for the study are tetracycline (30 μg), ampicillin (10 μg), chloramphenicol (30 μg), cotrimoxazole (25 μg), ciprofloxacin (5 μg), imipenem (10 μg), nalidixic acid (30 μg), cefotaxime (30 μg) (HiMedia, India). A fresh culture of isolates was grown in 5 ml of Mueller–Hinton (MH) broth up to turbidity of 0.5 McFarland standards and then it was spread using a sterile cotton swab on well-dried MH agar to prepare a lawn. After gentle air drying, the antibiotic discs were placed on the surface of the medium and incubated for 18–24 h at 37 °C for the appearance of a clear zone. The diameter of the zone of inhibitions around discs was measured and results were interpreted as sensitive or resistant.
Biofilm formation ability assay of Gram-negative pathogens
Biofilm-forming ability assay of the host S. typhimurium, V. cholerae, P. aeruginosa ADW44 and P. aeruginosa 27853(ATCC strain) was done using crystal violet assay (Kadouri & O'Toole 2005). Briefly, 190 μl of nutrient broth and 10 μl of bacterial culture were added to 96-well microtiter plates. Nutrient broth without bacterial culture was used as a control. Plates were incubated for 24 h at 37 °C. After incubation, wells with culture, as well as control were decanted and washed with 0.85% saline three times. Plates were allowed to dry at room temperature and 200 μl of 1% crystal violet was added to each well followed by 10–15 min of incubation. After incubation, wells are decanted again and washed with 0.85% saline three times. Plates are allowed to dry at room temperature for 15 min. Once dried, 200 μl of 33% glacial acetic acid was added and then transferred to a fresh plate. Absorbance was recorded at 630 nm using an ELISA plate reader and organisms were categorized as biofilm former or non-biofilm former.
Lytic activity the Bdellovibrio isolates
The lytic ability of the Bdellovibrio isolates was determined against four Gram-negative human pathogens, S. typhimurium, V. cholerae, P. aeruginosa ADW44 and P. aeruginosa 27853(ATCC strain). Optical density (OD) of the prey cells at a time interval of 24 h for 5 days was measured at 600 nm. To initiate the experiment, 1 ml each of Bdellovibrio lysate was inoculated into the prey cell culture with OD, OD 600nm = 1 in 50 ml dilute nutrient broth. The prey cell culture alone in 50 ml of dilute nutrient broth was kept as a control. The co-cultures of Bdellovibrio and prey along with the control were incubated at 30 °C with shaking at 120 rpm. The OD at 600 nm was determined using a spectrophotometer (UV-Vis Biospectrometer, Eppendorf) at 24-h time intervals for 5 days. This experiment was carried out for all 11 Bdellovibrio isolates on different prey cells in duplicates with similar conditions.
RESULTS
Isolation and identification of Bdellovibrio
Typical lytic plaques developed by Bdellovibrio on the lawn of Pseudomonas aeruginosa.
Typical lytic plaques developed by Bdellovibrio on the lawn of Pseudomonas aeruginosa.
Molecular identification of Bdellovibrio isolates
Representative agarose gel electrophoresis of samples for 16 s rDNA coding region. Lane M: 50-bp marker; Lanes 1, 2, 4, 5: positive samples; Lane 3: Negative sample.
Representative agarose gel electrophoresis of samples for 16 s rDNA coding region. Lane M: 50-bp marker; Lanes 1, 2, 4, 5: positive samples; Lane 3: Negative sample.
Representative agarose gel electrophoresis of samples for hit gene. Lane M: 50-bp marker; Lanes 1, 2, 3: representative samples.
Representative agarose gel electrophoresis of samples for hit gene. Lane M: 50-bp marker; Lanes 1, 2, 3: representative samples.
Representative agarose gel electrophoresis of samples for Bdellovibrio stolpii gene. Lane M: 50-bp marker; Lanes 1–11: samples.
Representative agarose gel electrophoresis of samples for Bdellovibrio stolpii gene. Lane M: 50-bp marker; Lanes 1–11: samples.
Antibiotic resistance and biofilm-forming ability assay of host bacteria
The details of the antibiotic susceptibility results of the test organisms are presented in Table 2. The zone of inhibition size is provided in the Supplementary Material (S2). Only Pseudomonas strains showed MDR pattern as it showed resistance to more than two classes of antibiotics. Biofilm-forming ability of the test results showed that the P. aeruginosa ADW44 is a strong biofilm-forming bacteria.
Antibiotic susceptibility pattern of the test organisms
Antibiotic . | Vibrio cholerae . | Pseudomonas aeruginosa ADW44 . | Pseudomonas aeruginosa 27853 . | Salmonella Typhimurium . |
---|---|---|---|---|
Tetracycline (30 μg) | S | R | R | S |
Nalidixic acid (30 μg) | I | R | R | S |
Chloramphenicol (30 μg) | S | R | R | S |
Ciprofloxacin (5 μg) | S | S | S | S |
Cotrimoxazole (25 μg) | S | R | R | R |
Cefotaxime (30 μg) | I | S | S | S |
Ampicillin (10 μg) | I | R | R | S |
Imipenem | S | S | S | R |
Antibiotic . | Vibrio cholerae . | Pseudomonas aeruginosa ADW44 . | Pseudomonas aeruginosa 27853 . | Salmonella Typhimurium . |
---|---|---|---|---|
Tetracycline (30 μg) | S | R | R | S |
Nalidixic acid (30 μg) | I | R | R | S |
Chloramphenicol (30 μg) | S | R | R | S |
Ciprofloxacin (5 μg) | S | S | S | S |
Cotrimoxazole (25 μg) | S | R | R | R |
Cefotaxime (30 μg) | I | S | S | S |
Ampicillin (10 μg) | I | R | R | S |
Imipenem | S | S | S | R |
Note: R, resistant; I, intermediate; S, sensitive.
Determination of lytic activity of Bdellovibrio strains
Graphical representation of lytic activity of Bdellovibrio isolates against Salmonella typhimurium: (a) activity of NBd1, YBd1 and RBd1; (b) activity of YBd2, YBd3, RBd2 and RBd3; (c) activity of KHBd1, KHBd2, KHBd3 and KHB4.
Graphical representation of lytic activity of Bdellovibrio isolates against Salmonella typhimurium: (a) activity of NBd1, YBd1 and RBd1; (b) activity of YBd2, YBd3, RBd2 and RBd3; (c) activity of KHBd1, KHBd2, KHBd3 and KHB4.
Graphical representation of lytic activity of Bdellovibrio isolates against V. cholerae: (a) activity of NBd1, YBd1 and RBd1; (b) activity of YBd2, YBd3, RBd2 and RBd3; (c) activity of KHBd1, KHBd2, KHBd3 and KHB4.
Graphical representation of lytic activity of Bdellovibrio isolates against V. cholerae: (a) activity of NBd1, YBd1 and RBd1; (b) activity of YBd2, YBd3, RBd2 and RBd3; (c) activity of KHBd1, KHBd2, KHBd3 and KHB4.
Graphical representation of lytic activity of Bdellovibrio isolates against Pseudomonas aeruginosa ADW44: (a) activity of NBd1, YBd1 and RBd1; (b) activity of YBd2, YBd3, RBd2 and RBd3; (c) activity of KHBd1, KHBd2, KHBd3 and KHBd4.
Graphical representation of lytic activity of Bdellovibrio isolates against Pseudomonas aeruginosa ADW44: (a) activity of NBd1, YBd1 and RBd1; (b) activity of YBd2, YBd3, RBd2 and RBd3; (c) activity of KHBd1, KHBd2, KHBd3 and KHBd4.
Graphical representation of lytic activity of Bdellovibrio isolates against Pseudomonas aeruginosa 27853: (a) activity of NBd1, YBd1 and RBd1; (b) activity of YBd2, YBd3, RBd2 and RBd3; (c) KHBd1, KHBd2, KHBd3 and KHBd4.
Graphical representation of lytic activity of Bdellovibrio isolates against Pseudomonas aeruginosa 27853: (a) activity of NBd1, YBd1 and RBd1; (b) activity of YBd2, YBd3, RBd2 and RBd3; (c) KHBd1, KHBd2, KHBd3 and KHBd4.
Figure 8 presents the predation of all 11 Bdellovibrio isolates against P. aeruginosa ADW44. Reduction in OD (OD600nm) after 24 h of incubation was observed when the P. aeruginosa ADW44 cultures were mixed with RBd1, YBd1, NBd1 isolates. The remaining Bdellovibrio strains YBd2, YBd3, RBd2, RBd3, KHBd1, KHBd2, KHBd3 and KHBd4 did not show much reduction in OD 600nm as compared with RBd1, YBd1, NBd1. Similar results were exhibited by these Bdellovibrio strains against other three tested organisms such as S. typhimurium, V. cholerae and P. aeruginosa 27853. But interestingly, the reduction in OD by YBd3 strain against V. cholerae did not match with the control. Overall, in the present study, three Bdellovibrio isolates namely RBd1, Ybd1 and NBd1 showed effective predation against all the tested organisms.
DISCUSSION
Antibiotic resistance in bacteria is a major concern in both national and international scenarios. Since the discovery of Bdellovibrio in 1962 (Stolp & Starr 1963), they are recognized as the predator species capable to prey Gram-negative bacteria which may impart a role in regulating microbial communities in environment (Caulton & Lovering 2020). The objective of the present study was to evaluate the lytic activity of Bdellovibrio isolated from untreated hospital wastewater against selected bacterial pathogens.
A total of eleven Bdellovibrio isolates were obtained from 30 untreated wastewater samples. Primary isolation of Bdellovibrio was performed using P. aeruginosa ADW44 as a prey organism. In another study, a total of 14 Bdellovibrio strains were obtained from the cultured fishponds using Aeromonas hydrophila J-1 as host (Chu & Zhu 2010). Lytic plaques were visible after 3–5 days of incubation and increased in size on further incubation. This phenotypic observation suggested that the predators were motile in nature within the soft agar. This observation clearly differentiates them from the phages (Feng et al. 2016). All 11 isolates of B. stolpii were obtained using the double-layer agar technique. This study supports the use of a double-layer agar plating technique and the use of dilute nutrient broth as an effective method for the isolation of Bdellovibrio strains from wastewater.
All the isolated Bdellovibrios in the present study were confirmed as B. stolpii by PCR. To the best of our knowledge, this is the first study to report the isolation of B. stolpii from hospital wastewater samples in India. Bdellovibrios have been previously isolated from soil, marine and rhizosphere, fresh water, brackish water, seawater, sewage water and animal intestine indicating their diverse prevalence in a terrestrial and aquatic environment but predominantly inhabiting B. bacteriovorus species (Medina et al. 2008).
The availability of genetic data to characterize Bdellovibrio is limited where most of the sequences in databases are 16S rDNA sequences used for phylogenetic treeing. In the present study, all 11 isolates were screened for 16S rDNA coding region using specific primers by providing desired amplicon size. Species-specific primers were used for confirmation of Bdellovibrio species. In the present study, the hit locus was successfully amplified in Bdellovibrio with an amplicon of two different sizes (Figure 4) which suggests that the isolates are different strains. Similar findings were obtained in another study by Oyedara et al. (2016). The presence of one genetic locus, hit is responsible for the transition from the attack phase to the growth phase in Bdellovibrio and the presence of this gene in isolates from our study strongly correlates with the study by Cotter & Thomashow (1992). The conversion of host-dependent strains of Bdellovibrio to saprophytic strains which can grow on heat-killed host bacteria is influenced by the mutations in the hit locus (Jurkevitch et al. 2000).
The activity of Bdellovibrio isolates was evaluated against four different clinical prey species such as P. aeruginosa ADW44, P. aeruginosa 27853, V. cholerae and S. typhimurium in dilute nutrient broth. In the present study, three Bdellovibrio isolates (RBd1, NBd1 and YBd1) exhibited good predatory behavior against the tested clinical isolates. Isolated strains of B. stolpii in the present study showed different killing rates among the tested clinical isolates. The predatory efficacy of YBd3 strain against V. cholerae in the present study is quite deviated when compared with the control. Similar findings were obtained in another study conducted by Rogosky et al. (2006), where differential predation by B. bacteriovorus 109J was observed when the predator cells were mixed and incubated with two types of prey cells. The observations drawn in the present study are also supported by other study findings where B. bacteriovorus UP readily preyed upon Gram-negative bacteria but not Gram-positive bacteria including the isolates that originated from the same sources (Feng et al. 2016). This finding has led to the conclusion that the selection of the appropriate predator for clinical purposes would require larger and more in-depth studies on predation to overcome predation resistance.
The inability of other remaining Bdellovibrio species to prey on other host cells could also be due to its structural features, which inhibit attachment, penetration or replication or may be due to the escape strategy of the host cell, which is not studied in the present study. Recent studies demonstrated applications of Bdellovibrio strains in biological wastewater treatment for reducing the burden of Gram-negative bacteria (Wu et al. 2019; Jafarian et al. 2020).
Bdellovibrio is able to prey on different clinical strains of a variety of beta-lactamases producing MDR Gram-negative bacteria regardless of their antibiotic resistance (Markelova 2010). In the present study, Figure 8 shows that P. aeruginosa ADW44, despite being the MDR, strong biofilm-forming bacteria was more susceptible to predation by B. stolpii than other prey cells used. YBd2, YBd3 and RBd2 effectively reduced the growth of the prey organism at the moment between 20 and 40 h. The obligatory parasitic lifestyle and predatory behavior make a way for their extensive use as biocontrol agents and an alternative to antibiotics in horticulture, aquaculture, livestock farming, food processing and medicine (Pérez et al. 2020). In the present study, selected clinical isolates of P. aeruginosa, S. typhimurium and V. cholerae were tested for predation. Studies also showed that Bdellovibrio is capable of preying on various other strains belonging to Acinetobacter, Escherichia, Bordetella, Aeromonas, Burkholderia, Pseudomonas, Citrobacter, Enterobacter, Klebsiella, Listonella, Morganella, Proteus, Vibrio, Salmonella, Yersinia, Shigella and Serratia genera (Bonfiglio et al. 2020). The ability of Bdellovibrio to lyse the MDR pathogenic organism P. aeruginosa in the current study indicates their potential applications in various fields.
One unique characteristic about Bdellovibrio that distinguish it from bacteriophage is its ability to invade the biofilm, suggesting it is better suited than phage to environments with multiple preys irrespective of their antibiotic resistance. The predatory efficacy of bacteriophages and Bdellovibrio comparison has not been carried out in the present study. Due to the predatory behavior of Bdellovibrio may replace the use of antibiotics in livestock feed which will eventually help to control antimicrobial resistance in human pathogens. Bdellovibrio as a strategy to combat antibiotic resistance gene transfer in wastewater treatment plants may help them to use as a novel technique to overcome horizontal gene transfer from clinical isolates to the environment which is yet to be addressed fully. Bdellovibrio may maintain its ability to reduce bacterial loads and preformed biofilms in clinical settings, as well as the environment. Further studies may prove their ability to target biofilm formation among various bacteria. Higher lytic activity toward Pseudomonas strains shows that these pathogens are more susceptible to predation by B. stolpii. The present study gives a predatory spectrum of B. stolpii against biofilm-forming P. aeruginosa ADW44 a wound isolate and underlines its potential use as a biocontrol agent which may prevent delayed wound healing.
CONCLUSIONS
Bdellovibrio strains isolated in the present study demonstrated the capability to prey upon selected Gram-negative pathogens such as P. aeruginosa, V. cholerae and S. typhimurium. The method developed in this study will allow a more rigorous assessment of the potential use of Bdellovibrio as a biocontrol agent versus biofilms. The differences in the prey range and amplification of hit locus of the isolates observed support the presence of heterogeneous groups of Bdellovibrio species in this region. This suggests further characterization and classification of Bdellovibrio into different subgroups by sequencing. More interestingly, predatory bacterium having multiple host ranges will have a great medical application. Studying their non-toxicity and other non-pathogenic characteristics on human cell lines may highlight their potential application as biocontrol agents to fight the infection in a broad approach as an antibiotic. However, multifaceted research studies are required to prove the advantages of the predatory bacterium to put into regular use in treating MDR infections. Due to the limited research on Bdellovibrio in India, this work is expected to form a base for further research on them with the potential biotechnological applications as the ultimate goal.
ACKNOWLEDGEMENTS
The authors are grateful to acknowledge Nitte University Faculty Research Grant [Sanction Order No: N/RG/NUFR1/NUCSER/2020/04 for providing financial support to D.M. The facilities provided for the study in Nitte University Center for Science Education and Research, Nitte (Deemed to be University) are gratefully acknowledged.
DATA AVAILABILITY STATEMENT
Data cannot be made publicly available; readers should contact the corresponding author for details.
CONFLICT OF INTEREST
The authors declare there is no conflict.