Isolation of soil bacteria able to degrade the anthelminthic compound albendazole

Chemicals and growth media

An analytical standard of ABZ (98%; Tokyo Chemical Industry^©, Zwijndrecht, Belgium) was used in media preparation and for analytical purposes. Analytical standard of ABZSO (98% purity) was also purchased from Tokyo Chemical Industry^© (Zwijndrecht, Belgium), while ABZSO₂ (97% purity) was purchased from Santa Cruz Biotech^© (Heidelberg, Germany). A mixture of ABZ, ABZSO and ABZSO₂ in acetonitrile (1,000 mg L⁻¹) were used for preparing serial dilutions ranging from 10–0.025 mg L⁻¹ which were used to construct calibration curves for residue quantification by HPLC.

Selective mineral salts media (MSM) and its nitrogen amended version (MSMN), supplemented with ABZ as the sole C and N or the sole C source respectively, were used for the isolation of ABZ-degrading bacteria. MSM and MSMN were prepared as described before (Karpouzas & Walker, 2000). Growth media were spiked with a 5,000 μg ml⁻¹ filter-sterilized solution of ABZ in DMSO (Molecular Biology Grade; Sigma Aldrich^©, St. Louis, MI, USA) aiming to a final concentration of 5 μg ml⁻¹ of ABZ in the medium. This concentration was the highest tested concentration of this compound that could be fully dissolved in the aqueous growth medium without any solubility issues and precipitation. DMSO levels in the medium never exceeded 0.1%. Growth media were also amended with 0.05% of Tween 20 to enhance ABZ solubility, as suggested in our earlier studies (Lagos et al., 2021). Agar plates of the aforementioned media plus ABZ and Tween 20 were prepared by addition of 15 g L⁻¹ agar.

Enrichment cultures and isolation of ABZ-degrading bacteria

To isolate ABZ-degrading bacteria, we employed enrichment cultures in MSM and MSMN supplemented with ABZ. A soil from a livestock unit in Lesvos Island, Greece, 39°16′21.4″N 26°15′55.7″E, with history of ABZ administration and high degradation capacity towards ABZ (Lagos et al., 2022) was used for bacteria isolation. Prior to the onset of the enrichment cultures, the soil was repeatedly treated with ABZ (5 μg g⁻¹), three times on 15-day intervals to stimulate and activate the microbial community able to degrade ABZ. This concentration level is higher than the concentration levels of ABZ often detected in soils (Thiele-Bruhn, 2003; Liebig et al., 2010; Navrátilová et al., 2023) but it was selected based on (i) previous soil studies which have indicated that the same soil was able to degrade ABZ concentrations of 2 mg/kg or higher at an accelerated mode (Lagos et al., 2023) and (ii) on previous soil studies with pesticides and other organic pollutants that have indicated that such concentration levels are essential to provide the necessary carbon and energy to stimulate the specialized pollutant-degrading soil microbiota (Topp et al., 2013; Rousidou et al., 2016; Lagos et al., 2019). After completing the pre-treatment, 0.5 g of soil were used to inoculate triplicate bottles per medium (20 ml), while duplicated non-inoculated samples containing the same volume of each medium were used as abiotic controls. All cultures were incubated in an orbital shaker in the dark at 25 °C. The degradation of ABZ was measured by analyzing samples at regular interval by HPLC as described below. At the point where degradation of ABZ was >70% an aliquot of each culture (0.5 ml) was transferred in fresh triplicate cultures. The same procedure was repeated for four cycles in total and at the point of 65–70% degradation of ABZ in the fourth enrichment cycle, a serial dilution was prepared and spread on MSM or MSMN agar plates amended with ABZ (5 μg ml⁻¹). The plates were then placed for incubation at 25 °C. After 3–4 days of incubation growing colonies were selected and transferred in the corresponding liquid media. The capacity of the selected colonies to degrade ABZ was determined at 7 days via HPLC. Aliquots of cultures which showed a high degradation capacity (>70% degradation in 7 days) were sub-cultured in fresh liquid media in triplicates, to confirm their degradation capacity. Only cultures exhibiting >60% degradation in 7 days were considered as positive and they were all derived from MSMN. The selected cultures were plated on MSMN + ABZ agar plates to check purity. They were then processed for DNA extraction, and further molecular analysis as described below.

Albendazole residue analysis

ABZ was extracted from liquid media by mixing 0.5 mL of culture with 0.5 mL of acetonitrile. The mixture was vigorously vortexed for 30 sec, then filtered through 0.45-μm PTFE hydrophobic syringe filters and directly analyzed in a Shimadzu HPLC–DAD system equipped with a Grace Smart RP C18 column (150 mm × 4.6 mm) (Shimadzu Corporation, Kyoto, Japan) as described before (Lagos et al., 2022). Briefly, ABZ, ABZSO and ABZSO₂ were eluted using a gradient mobile phase of 30:70 acetonitrile:water (v/v) + 0.1% H₃PO₄ and they were detected at 227 nm. Fortification tests at three concentration levels (0.1, 1 and 10 mg L⁻¹) showed mean percentage recoveries for ABZ, ABZSO, ABZSO₂, of 91.7%, 90.4%, 95.2% (CV < 11.3%) respectively. The LOD and LOQ values for all analytes were 0.02 and 0.05 mg L⁻¹ respectively. The concentrations of ABZ and its oxidation products were determined using an external calibration curve by injection of matrix-matched standard solutions (0.02–10 mg L⁻¹) of a mixture of all analytes. At this concentration range calibration curves showed high linearity with r² > 0.99. The repeatability of the method for all three analytes, expressed as the relative standard deviation of the recovery at all fortification levels was acceptable (≤9.4%). The reproducibility of the method for all three analytes, determined as the % recoveries obtained every 10 days for a 30-day period for aqueous matrices (minimal media) fortified at the concentration of 1 mg L⁻¹, was also acceptable (≤12.9%).

Molecular identification of the ABZ-degrading bacteria

Bacteria DNA extraction was performed with the Nucleospin® Tissue kit (Marcherey–Nagel, Düren, Germany). Briefly, the near full-length (1,500 bp) 16S rRNA gene of bacterial cultures was amplified with primers 8f–1512r (Felske et al., 1997) as described by Perruchon et al. (2016). The identity of the isolated bacteria was determined via cloning the PCR products, using the pGEM®-T easy plasmid vector, and sequencing of the full length 16S rRNA gene. Three clones for each isolate were Sanger sequenced and the obtained sequences were edited manually and analyzed for best match with the Basic Local Alignment Search Tool (BLAST, v.2.9.0) (Altschup et al., 1990). The closest relatives obtained plus an outgroup sequence were aligned with the Muscle software (Notredame, Higgins & Heringa, 2000). Uninformative blocks and misalignments were removed with the GBlocks software (Talavera & Castresana, 2007), and the sequence alignment obtained was utilized for the construction of maximum likelihood trees generated according to the general time reversible model, with gamma rate heterogeneity and accounting for invariable sites, using the PhyML software (v.3.1) (Guindon & Gascuel, 2003). The sequences of the clones which studied were submitted in GeneBank NCBI, database with the accession numbers OP604271 to OP604273.

Enrichment cultures in MSM and MSMN media

The degradation of ABZ in enrichment cultures in MSM and MSMN is presented in Fig. 1. In the first enrichment cycle in both growth media degradation of ABZ was over 70% after 6 days. In both growth media and across enrichment cycles, the degradation of ABZ showed similar patterns with over 60% and 70% degradation of ABZ in MSM and MSMN respectively after 6 days from the start of each enrichment cycle (Figs. 1A and 1B). Abiotic degradation of ABZ in the non-inoculated controls in both media never exceeded 20% (Fig. 1), suggesting that the degradation of ABZ observed in the inoculated cultures is microbially driven. ABZSO (main transformation product of ABZ) and ABZSO₂ were detected in both inoculated and non-inoculated cultures at low levels suggesting that their formation was mostly due to abiotic degradation process, as also suggested by earlier studies (Liou & Chen, 2018). However, the contribution of biotic processes in the formation of ABZSO and ABZSO₂ cannot be entirely excluded, especially in the MSM where slightly but not significantly higher amounts of ABZSO were formed in the inoculated vs non-inoculated cultures from the second enrichment cycle onwards. Still, it is important to note that the sum of these oxidative derivatives never exceeded 15% of the initial amount of the parent compound implying the formation of other transformation products during the biotic degradation of ABZ that were not monitored in our study. Similar studies with other xenobiotic compounds carrying thioether moieties in their molecule, like the pesticide fenamiphos, showed that soil bacteria were actively degrading fenamiphos and its sulfoxide and sulfone derivatives through hydrolysis rather than oxidation (Caceres et al., 2009; Chanika et al., 2011). We speculate that a similar pathway might be also active in our enrichment cultures where ABZ itself and its oxidation derivatives, ABZSO and ABZSO₂, are hydrolyzed through removal of the methyl carbamic moiety of the benzimidazole ring to 2-amino inactive derivatives, which were not monitored in our study and also in other relevant earlier studies. However, this speculative transformation pathway of ABZ should be further verified in follow up shotgun metabolomic analysis.

Isolation and screening of Albendazole-degrading bacteria

After completion of enrichment cultures and plating, 20 and 12 morphologically distinct bacterial colonies were selected from MSMN + ABZ and MSM + ABZ agar plates respectively and screened for their ability to degrade ABZ (Figs. 2 and 3). Four colonies which showed more than 50% degradation, were selected from MSM cultures (Fig. 2A) but their degrading capacity was not verified in a second round of cultivation and testing (Fig. 2B). In case of MSMN, five colonies which presented >60% degradation of ABZ after 7 days of incubation were selected for further testing (Fig. 3A). From these only two cultures, named C3 and C13, maintained their high degradation capacity and exhibited >60% degradation of ABZ after 7 days of incubation (Fig. 3B). ABZ was partially transformed to ABZSO in both inoculated and non-inoculated cultures, while small amounts of ABZSO₂ were also detected but only in the inoculated cultures. Previous studies with fungal and bacterial isolates tested for their degrading capacity against ABZ also identified ABZSO and ABZSO₂ as the sole transformation products of ABZ (Prasad, Girisham & Reddy, 2009, 2010). In contrast Prasad et al. (2008) observed, besides ABZSO and ABZSO₂, the formation of a new N-methylated derivative, produced by the degradation of ABZ by a Cunninghamella blakesleeana fungal strain.

Identification of ABZ-degrading bacteria

Based on their degradation capacity against ABZ the two isolates were further identified via molecular means. Clone libraries, prepared from cultures C3 and C13, revealed that the phylotypes represented in these cultures showed highest sequence match to the 16S rRNA gene sequence of bacteria of the genus Acinetobacter. Phylogenetic analysis based on the full-length 16S rRNA gene sequence verified the assignment of the two bacterial isolates to the genus Acinetobacter. Specifically, clones from culture C3 grouped with species Acinetobacter oleivorans and Acinetobacter calcoaceticus, while clones obtained from culture C13 were phylogenetically closer to Acinetobacter pittii (Fig. 4). However, the low bootstrap values does not allow the assignment of the two isolates to the species level. Bacteria of the genus Acinetobacter are ubiquitous in soil and they are characterized as metabolically versatile bacteria able to catabolize a wide range of natural compounds, implying active participation in nutrient cycling (Jung & Park, 2015). They are also known as efficient degraders of xenobiotic aromatic compounds like phenolic derivatives, quinones, pyridines, indoles (Paller, Hommel & Kleber, 1995; Ying et al., 2007; Zhang et al., 2021) and pesticides. For example, A. calcoaceticus and A. oleivorans strains were able to degrade the insecticide fipronil (Uniyal et al., 2016), while Zhan et al. (2018) and Singh, Suri & Cameotra (2004) isolated Acinetobacter stains able to degrade pyrethroids and atrazine respectively.

To date there are a few reports of microorganisms able to degrade ABZ. Jin et al. (2013) isolated a Rhodococcus strain that was able to degrade ABZ and use it as a C source, in agreement with our Acinetobacter isolates that degraded ABZ only in MSMN where the AH served as a sole C source. Prasad, Girisham & Reddy (2010) screened several bacterial strains for their capacity to oxidize ABZ to ABZSO and identified Enterobacter aerogenes, Klebsiella aerogenes, Pseudomonas aeruginosa and Streptomyces griseus stains as active degraders of ABZ. Besides bacteria, Prasad et al. (2008) and Prasad, Girisham & Reddy (2009) also isolated fungal degraders of ABZ like a Fusarium moniliforme strain and a Cunninghamella blakesleeana strain.