Isolation and characterization of entomopathogenic bacteria from soil samples from the western region of Cuba

Authors


ABSTRACT:

The use of insect pathogens is a viable alternative for insect control because of their relative specificity and lower environmental impact. The search for wild strains against dipterans could have an impact on mosquito control programs. We have made an extensive screening of soil in western Cuba to find bacteria with larvicidal activity against mosquitoes. A total of 150 soil samples were collected and isolates were identifying using the API 50 CHB gallery. Phenotypic characteristics were analyzed by hierarchical ascending classification. Quantitative bioassays were conducted under laboratory conditions following the World Health Organization protocol in order to ascertain the toxicity and efficacy of isolates. The protein profiles of the crystal components were determined by SDS-PAGE. Eight hundred and eighty-one bacterial isolates were obtained, and 13 isolates with entomopathogenic activity were isolated from nine samples. Nine isolates displayed higher entomopathogenic activity against both Cx. quinquefasciatus and Ae. aegypti compared with the reference strain 266/2. All toxic isolates showed higher biological potency than the 266/2 strain. These isolates with high entomopathogenic activity displayed a protein pattern similar to the B. thuringiensis var. israelensis IPS-82 and 266/2 strains. These results are a valuable tool for the control of Diptera of medical importance.

INTRODUCTION

Chemical insecticide releases have led to the emergence and spread of resistance in vectors of human diseases and to environmental degradation (Ibarra et al. 2003). The use of insect pathogens is a viable alternative for vector control because of their relative specificity and lower environmental impact. Diverse formulations containing bacterial agents such as Bacillus thuringiensis var israelensis and Bacillus sphaericus have been developed for mosquito control (Amadio et al. 2009, Cetin et al. 2007). Several collections of B. thuringiensis isolates have been well characterized worldwide. The strains have been isolated from varied microhabitats such as soil, plant leaves, dead insects, and stored grain, as well as aquatic environments such as marine sediments, mangroves, and fresh water (Ibarra et al. 2003, Armengol et al. 2007).

Research to identify new pathogenic strains is the first step in the development of alternatives to current methods of vector control. Consequently, the aim of the present study was to carry out the first extensive screening of soil bacteria in Cuba in order to select bacterial isolates with larvicidal activity against the mosquitoes Aedes aegypti and Culex quinquefasciatus.

MATERIALS AND METHODS

Reference strains

Bacteria: International Standard IPS-82 (Pasteur Institute, France) and 266/2 strain of Bacillus thuringiensis var. israelensis from the commercial formulation Bactivec (LABIOFAM, Cuba) were used.

Mosquitoes: Aedes aegypti (Rockefeller strain) and Culex quinquefasciatus (Quibú strain, Havana). Adult mosquitoes were maintained at 27 ± 2° C with 85% RH and 12:12 h L:D regime in collapsible cages (BioQuip, U.S.A.) supplemented with 5 ml of one-day-old tap water and 10% sugar-saturated cotton on the top of the cage. Blood-feeding was allowed on guinea pigs. Larvae were reared in a sparse density (∼250 per 45×35 cm tray) with daily supplements of fish food sprinkled on the water surface.

Soil samples

Soil samples were collected in glass tubes from the surface to a depth of 5 cm. Samples came from urban areas, cultivated fields, woodlands, wetlands, and coastal ecosystems. A total of 150 soil samples were collected in the western region of Cuba from October, 2008 to March, 2009. The locations of the collection points were geo-referenced using a global positioning system.

Growth conditions and bacterial identification

One gram of soil was cultured in 10 ml of nutrient broth medium for 24 h at 30° C with orbital shaking (Retomed, Cuba) at 150 rpm. An aliquot of cultured liquid was spread on nutrient agar plates at 30° C for 24 h. Bacterial colonies were isolated on nutrient agar plates 24 h at 28° C. Gram staining and observation under a phase contrast microscope were carried out.

The Gram positive sporulated rods were identified following the algorithm described by Parry et al. (1983). The fermentation and assimilation of carbohydrates and their derivatives was performed using the API, 50CHB gallery (BioMérieux, France). The parasporal inclusion observation by phase contrast microscopy was used as a criterion for identification of B. thuringiensis.

Antimicrobial susceptibility tests

Antimicrobials: Oxacillin, gentamicin, ciprofloxacin, levofloxacin, erythromycin, clindamycin, quinupristin-dalfopristin, linezolid, teicoplanin, vancomycin, minocycline tetracycline, rifampin.

Broth microdilution susceptibility tests were performed with standard procedures. Briefly, the drugs were diluted in cation-supplemented Mueller-Hinton broth, and the inoculum was 5 × 105 CFU/ml. The minimal inhibitory concentration (MIC) was recorded after 24 h of incubation at 35° C. Interpretation of the MIC values followed CLSI standards (Clinical and Laboratory Standards Institute (CLSI 2005).

Assays for hemolytic toxins

Hemolysis assays were performed in microtiter plates using sheep blood, and erythrocytes were diluted in round-bottom-well microdilution plates (Nunc) in 100 μl of 50% (vol/vol) PBS-0.05% (wt/vol) gelatin-2.25% (wt/vol) glucose.

Bacillus thuringiensis index

The index was obtained by the ratio of the number of B. thuringiensis colonies isolated in relation to the total number of colonies of Bacillus (Carreras 2009).

Bacterial formulations

The fermentation medium consisted of sucrose, bacteriological peptone, yeast extract, and inorganic salts (12.5 mmol/L MgSO4; 0.05 mmol/L MnSO4; 1.2 mmol/L FeSO4; 1.2 mM ZnSO4; 25 mmol/L CaCl2) incubated at 30° C 48–72 h at 150 rpm (Retomed, Cuba), until the formation of large amounts of spores and parasporal bodies occurred. Concentrations were expressed in mg/ml (dry weight).

Larvicide efficacy

Preliminary qualitative bioassays were achieved by adding 500 μl of the bacterial suspensions to 50 ml of tap water containing 25 mosquito larvae. Mortality was recorded at 24 h. The isolates were classified as toxic when mortalities were up to 50% (Chaves et al. 2008).

Quantitative bioassays were conducted following the World Health Organization (WHO) protocol. Twenty-five larvae were introduced into 120 ml cups with 100 ml of tap water. Four replicates per dose were included and the experiments were repeated at least three times. Five concentrations with mortalities between 10% and 90% were accepted for validating the bioassay. Mortality data were utilized to calculate the lethal concentrations for 50% and 90% of exposed individuals (LC50 and LC90, respectively) through log probit analysis. These values were compared to those obtained for the reference strains in order to estimate the efficacy for each isolate. The means of larval mortality caused by each isolate against both Cx. quinquefasciatus and Ae. aegypti were calculated. Data were analyzed by multifactor analysis of variance and analyzed a posteriori by Tukey HSD mean multiple comparison test. A value of p<0.05 was considered statistically significant. The LC95/ LC50 ratio was calculated and used as an expression of the efficiency of a formulation (Osborn et al. 2007). The potency (ITU/mg) was evaluated following procedures described by WHO compared with the International Standard IPS-82 (15000 ITU/mg) for titration against laboratory-reared Ae. aegypti larvae. A series of bioassays was carried out with five repetitions for each of six different concentrations tested and one untreated control.

Phenotypical characterization

A binary data matrix was constructed containing results of biochemical tests, antimicrobial susceptibility, hemolysis, colony growth type, and larvicidal activity. Statistical analysis was performed by hierarchical ascending classification (HAC). Parameters were proximity type (similarity), agglomeration method (Jaccard), clustering method (medium link), and truncation level (0.03).

Protein profiles

The bacterial isolates were inoculated in tubes containing 5 ml of nutrient broth supplemented with inorganic salts at 28° C at 300 rpm agitation until complete sporulation and crystal formation occurred. The suspensions were centrifuged (10,000 g for 20 min) and the pellets were washed twice with 1 mol/L NaCl and then with distilled water. The pellet was resuspended in 100 μl of distilled water and 100 μl of sample buffer (mmol/L Tris-HCl 500 pH 6.8, 10% SDS, 4% 2-mercaptoethanol, 8% glycerol, 0.1% bromophenol blue), and boiled at 100° C for 6 min (Carreras et al. 2004).

The protein profiles of the crystal components were determined by sodium dodecyl sulfate (SDS)-poliacrilamide gel electrophoresis (PAGE) (Schägger and Von Jagow 1987) with 10% acrylamide separating gels. Ten μl of each sample were loaded onto a gel immediately before electrophoresis. Five μl of a molecular weight marker (Broad Range Protein Molecular Weight Markers, Promega, U.S.A.) was added to each gel. The images obtained from the gels were processed and the molecular weight of each protein was calculated with GelQuant version 2.7.0 software (Bio-Imaging Systems, Israel). The protein patterns were ranked by ascending hierarchical classification by the statistical system NCSS 2007, with the following parameters: proximity type (dissimilarity), agglomeration method (Jaccard), clustering method (medium link), and truncation level (0.5). The results were plotted on a dendrogram.

RESULTS

One hundred-fifty soil samples from the western region of the Cuban archipelago (Pinar del Rio, Havana City, Havana Province, and Matanzas) were collected. A total of 881 bacterial isolates were obtained: 773 were Gram-positive sporulated and 108 Gram-positive not sporulated bacilli. The most frequently isolated bacterial species were B. cereus (53.8%) and B. thuringiensis (29.5%); both were isolated from all sampled provinces (Figure 1). The highest diversity of species was found in Matanzas province (B. cereus, B. thuringiensis, B. anthracis, B. mycoides, B. megaterium, non-spore forming Gram-positive bacilli, B. stearothermophilus, and B. lentus). Likewise, the higher B. thuringiensis index (0.39) was recorded at Pinar del Rio and Havana Province. The B. thuringiensis index of Havana City was 0.35, while Cayo Largo del Sur had the lowest index of Bacillus thuringiensis (0.1). Microdilution susceptibility testing of toxic isolates revealed that all strains were susceptible to gentamicin, ciprofloxacin, levofloxacin, moxifloxacin, clindamycin linezolid, teicoplain, vancomycin, minocycline, and rifampin. All isolates were resistant to oxacillin (Table 1).

Figure 1.

Collected points positive for B. thuringienisis (triangles). Samples with larvicidal activity against Ae aegypti and/or Cx quinquefasciatus (diamonds).

Table 1.  Antimicrobial resistance patterns and intermediate susceptibility in all the isolates with larvicidal activity.
Antimicrobial resistance patternsIntermediate susceptibilityIsolates
Oxacillin266/2, M29, R84, U81, X48, X23
OxacillinQuinupristin-dalfopristinIPS82, R85
Oxacillin, tetracyclineErythromycinL95
Oxacillin, erythromycinL910, O89
Oxacillin, quinupristin-dalfopristinR87, R89, M910, T39

The preliminary qualitative bioassays showed 13 isolates of B. thuringiensis with toxicity against mosquito larvae (Table 2). The isolates with entomopathogenic activity were obtained from nine samples with high predominant phaeozems soils. Single ovoid parasporal crystals were observed in all these isolates, similar to the reference strains of serotype H-14. Hemolytic activity was detected in most of the isolates (Table 2).

Table 2.  Soil types (FAO 2003), hemolytic activity, efficacy, and potency of isolates.
IsolatesSoils typeHemolytic activity% larval mortalityITU/mg
Culex quinquefasciatus Aedes aegypti
L95Phaeozems+1001002118
L910Phaeozems+1001001203
M29Phaeozems+1001002400
M910Ferralsols+060
O89Gleysols+600
R84Phaeozems+100100916
R85Phaeozems+100100318
R87Phaeozems+100100845
R89Phaeozems+100100497
T39Ferralsols6060
U81Phaeozems+1001001948
X23Kastanozems+600
X48Kastanozems+1001001117
266/2 strainReference strain+10010062

Nine isolates displayed higher entomopathogenic activity against both Cx. quinquefasciatus (Figure 2) and Ae. aegypti (Figure 3) compared with the reference strain 266/2 (ANOVA, p <0.05). On the basis of both larvicide activity and efficiency, the most active isolate against Cx. quinquefasciatus was L910 and R84 against Ae. aegypti.

Figure 2.

Entomopathogenic activity of isolates of B. thuringiensis on Cx. quinquefasciatus. Markers connected by lines represent lethal concentrations 10, 50, and 90 plotted on the vertical axis primary. No line markers represent the relationship LC95/LC50, plotted on the secondary vertical axis.

Figure 3.

Entomopathogenic activity of isolates of B. thuringiensis on Ae. aegypti. Markers connected by lines represent lethal concentrations 10, 50 and 90, plotted on the vertical axis primary. No line markers represent the relationship LC95/LC50, plotted on the secondary vertical axis.

The toxic isolates obtained in this study exhibited higher biological potency than the 266/2 strain. The M29, L95, and U81 isolates had the highest values that were significantly different from the rest. The R85 isolate had the lowest biological potency, but this value was six times higher than obtained with strain 266/2 (Table 2). The HAC analysis data matrix of biochemical tests, antimicrobial susceptibility, hemolysis, colony growth type, and larvicidal activity grouped the isolates in five HAC classes. Most isolates (L910, M29, R84, R85, R87, R89, U81, and X48) and both reference strains were included in the same class. X23 and O89 isolates were incorporated in another class and the remaining isolates (L95, T39, and M910) were included in individual classes.

The nine isolates with highest entomopathogenic activity showed a similar protein pattern to the IPS-82 and 266/2 strains of B. thuringiensis var. israelensis. A Cry 4 protein family was observed at a level of 127–135 kDa (Bravo et al. 2007). The protein band of 78 kDa corresponding to Cry10 is often produced in low levels and was poorly visualized in our study (Thorne et al. 1986). The proteins observed between 66 and 72 kDa have been described as family Cry 11 (Berry et al. 2002, Bravo et al. 2007). In all isolates and reference strains, a band at the level of 27 kDa corresponding to Cyt 1A (Manceva et al. 2005) was visualized. The M910, X23, T39, and O89 isolates lacked the protein bands corresponding to Cry 10 and Cry 11 proteins. Of this group, only M910 showed a Cry 4 protein.

The HAC of isolates based on their protein profiles allowed grouping isolates into four clusters (Figure 4):

Figure 4.

Dendrogram of dissimilarity based on the patterns obtained from the protein profile of isolates of B. thuringiensis from soil samples with entomopathogenic activity.

  • 1L95, L910, M29, R84, R85, R87, R89, U81, and X48: bands of 135, 78, and 66 kDa. The reference strains IPS-82 and 266/2 were included in this class, with a distance of 0.9 with respect to other clusters.
  • 2T39 and X23: bands of 170 and 42 kDa.
  • 3M910: bands of 135 and 42 kDa.
  • 4O89: 36-kDa band.

A band of 98 kDa was observed in clusters 2, 3, and 4.

DISCUSSION

Bacillus thuringiensis is a highly diverse species; its isolates have been recovered from various environments. The isolation of native B. thurigiensis strains in Latin-America has been reflected in the high variety of climatic zones in this geographical area (Ibarra et al. 2003). There are reports of extensive isolation of the Bacillus genus in Cuba but these focused on the control of agricultural pests (Carreras 2009). In a previous short screening, three Cuban native strains with activity against Diptera of medical importance were isolated (González et al. 2011). The present study was oriented to isolate enthomopathogenic bacteria against vectors of human illness also, but a higher number of samples from the western region of the Cuban archipelago were processed.

The high percentage of strains of the genus Bacillus was influenced by the methodology, which focused on the production of entomopathogenic bacteria. This included the sample type, its collection in the shade to avoid UV light damage and high temperature, the culture medium, and incubation conditions (Parry et al. 1983). The soils of Cuba are an important source of B. thuringiensis. We obtained values of the B. thuringienisis index similar to those obtained by Carreras (2009). Isolates with more larvicidal activity were isolated from phaeozems soils. The western region of Cuba has lateritic soils dominated by minerals, with free iron content not exceeding 3% and pH under 7. These conditions could play a role in the persistence of the spores of B. thuringiensis (Maduell et al. 2008, Du et al. 1994, Carreras 2009). All these results support those obtained in our study. Therefore, it explains the lower rate of isolation of B. thuringiensis at Cayo Largo del Sur, which has typical coastal soils (sandy, oolitic, and carbonate) (López 2000, Ortega and Arcia 1986).

Although many aspects of the structure and mode of action of B. thuringiensis have been studied in detail, its ecology is not well established (Raymond et al. 2010). The ecological niche of the organism and its success as an entomopathogen varies in natural conditions. It appears that it is a soil bacterium with incidental insecticidal activity (Martin and Travers 1989) that may provide symbiotic protection against insect attack (Smith and Couche 1991) and may belong to the intestinal microbiota of several insect orders (Jensen et al. 2003). Obviously, B. thuringiensis is closely related to the soil, and the high resistance of its spores to adverse environmental conditions makes the isolation of these samples possible, which has the advantage of easy conservation and transportation.

In the laboratory, L95, L910, M29, R84, R85, R87, R89, X48, and U81 isolates showed higher entomopathogenic activity against larvae of Cx. quinquefasciatus and Ae. aegypti than did the reference strain 266/2, which has been applied in Cuba for mosquito control (González et al. 2011). In all these isolates a similar pattern protein was observed, with the major proteins of 135, 78, 66, and 27 kDa. According to several studies, this combination is extremely effective against insects of the Order Diptera (Perez et al. 2005, Ibarra et al. 2003).

Protein profiles by SDS-PAGE from bacterial cell lysates have been used routinely for the differentiation and characterization of isolates of B. thuringiensis in order to determine the main entomopathogenic factors (Sreshty et al. 2011, Ibarra et al. 2003). Cry4 protein (127–135 kDa) has great pathogenic variability with activity against dipterans, nematodes, mites, and Lepidoptera (Mena et al. 2003, Howlader et al. 2010). The 28 kDa band corresponding to Cyt 1A has detergent activity (Manceva et al. 2005). This protein was present in all isolates with entomopathogenic activity, including those with LC50 higher than the reference strain, evidence that by itself is not responsible for the high pathogenicity of B. thuringiensis against Ae. aegypti (Boisvert and Boisvert 2000).

The 78 kDa band that corresponds to Cry10 protein (Thorne et al. 1986) was poorly visualized in our study. This is consistent with other reports that describe their low expression in isolates of B. thuringiensis var. israelensis (Hernandez-Soto et al. 2009). Finally, the Cry proteins belonging to group 11 are highly toxic against Diptera and also have activity against Lepidoptera and Coleoptera (Tailor et al. 1992, Armengol et al. 2007), reported to be between 66 and 72 kDa (Berry et al. 2002). The absence of these two protein bands in the isolates M910, X23, T39, and O89 could explain their lower larvicidal activity.

Analysis of the relationship between protein profile and entomopathogenic effectiveness in the isolates and reference strains shows that all these proteins have toxic action and its efficiency depends on their combination (Boisvert and Boisvert 2000). Cyt1A is synergistic with Cry11, acting as a receptor for binding to the membrane of intestinal cells, but the molecular mechanism has not been elucidated (Perez et al. 2005, Tabashnik 1992). We also noted a synergy between Cry10A and Cyt1A (Hernandez-Soto et al. 2009), and between Cyt1A and Cry4, but not between Cry4 and Cry11A (Tabashnik 1992). The distribution of some bacteria is largely related to geographic, climatic zones, and human and insect population movements. Spatial distribution helps us understand the evolution and transmission through the analysis of bacterial populations. GPS tracking increases the accuracy of isolation studies. An understanding of the dynamics of bacterial populations can help to determine appropriate interventions, including vector control (Baker et al. 2010).

Isolates with higher efficiency and effectiveness as entomopathogens were obtained from three municipalities of Havana City and from a locality of Pinar del Rio province (Las Terrazas). In both cases, three factors might have been favored: soil type (soils with high organic matter accumulation), the presence of hosts and application of biological pesticides based on B. thuringiensis var. israelensis. Havana City, as a large human settlement, has conditions favorable for the development of Cx. quinquefasciatus and Ae. aegypti. Pinar del Rio isolates were obtained from samples from rural areas with high infestation with Simuliidae that were also susceptible to B. thuringiensis var. israelensis.

Vilas-Boas and Lemos (2004) obtained similar results in Brazil, where B. thuringiensis isolates were associated with the habitats of their hosts. These authors considered that biotic environmental factors could influence the presence of microorganisms in soil, similar to abiotic factors such as pH, temperature, moisture, oxygen, nutrients, and soil texture. Bacterial ecology studies using manipulated field trials showed that the presence of host insects significantly increased the abundance of bacteria in group B. cereus, both in terms of numerical population size and the proportion expressing the cry genes (Raymond et al. 2010). We speculate that the synthesis of Cry toxins allow B. thuringiensis to promote their own ecological niche to kill insects and use their bodies to grow. The natural conjugation between B. thuringiensis and B. cereus occurs with high frequency (Santos et al. 2010). Logically, one would expect that in places with high densities of insect hosts, the related bacteria populations have the plasmids that confer entomological pathogenicity.

Similarly, the application of a biological product based on B. thuringiensis affects the population of the B. cereus group (Raymond et al. 2010). The plasmid transfer by conjugation carrying cry genes could promote genetic diversity of wild populations. Even when the bacterium is applied as part of a massive vector control product, it has been observed that their final destination is the soil as spores, since the vegetative bacterial cell cannot divide (recycle) or maintain entomopathogenic activity for a long period (Boisvert and Boisvert 2000, Hendriksen and Hansen 2002).

On the other hand, the bacterial strain from the biolarvicide, which has been artificially maintained for large periods, finds in nature the ideal conditions for biological growth, including the expression of cry genes. The larvicide Bactivec has been released in Havana City for mosquito control, and in the community Las Terrazas has been applied experimentally for black fly control. Isolates could correspond to natural transformations of wild strains with the 266/2 strain, which reinforce their natural mechanisms of gene expression, especially those contained in the large plasmids. This would explain the significantly higher entomopathogenic activity of the isolates with the same protein profile.

The larvicidal efficacy and biological potency determined for the isolates L95, L910, M29, and U81 suggests they are promising for the active principle of new formulations, since the results obtained in bioassays are better than those found for the strain 266/2. However, phenotypic criteria currently are not enough, and the analysis of a large number of variables and observations bringing an array of solid data from the statistical point of view is an aspect to consider. Therefore, considering the particular phenotypic characteristics and high entomopathogenic activity, isolate L95 was considered to be the best candidate for the development of new biological larvicides.

Ancillary