Isolation and identification of guaiacol producing Alicyclobacillus fastidiosus strains from orchards in Poland.

The genus Alicyclobacillus comprises a group of Gram-positive, thermo-acidophilic bacteria that are capable of producing highly resistant endospores during unfavorable environmental conditions. The members of this genus inhabit natural environments, including hot springs and soils. The main reason behind the spoilage of final commercial fruit products by Alicyclobacillus is the contamination of fruits with soil at the time of harvesting. Some of the Alicyclobacillus species, including Alicyclobacillus acidoterrestris, are categorized as spoilage bacteria due to their ability to produce off-flavor compounds (e.g., guaiacol and halophenols) that adversely affect the taste and aroma of beverages. In our study, Alicyclobacillus species were isolated from Polish orchard soils and fruits and were subjected to 16S rDNA sequencing. The results of the analysis showed that the isolated strains belonged to A. acidoterrestris and Alicyclobacillus fastidiosus species. All the three isolated strains of A. fastidiosus (f1, f2, f3) exhibited similar morphological and biochemical properties as the strain described in the literature. However, these isolated strains were able to produce guaiacol at temperatures of 20°C, 25°C, and 45°C. Thus, the strains of A. fastidiosus discovered in the present study can be included in the group of spoilage species as they possessed the gene responsible for the production of guaiacol.


INTRODUCTION
The genus Alicyclobacillus comprises Gram-positive, thermo-acidophilic, nonpathogenic, and spore-forming bacteria. Alicyclobacillus species possess unique fatty acids (cyclohexane or cycloheptane) in their cell membrane (Deinhard et al., 1987a;Deinhard et al., 1987b). These thermo-acidophilic bacteria were first isolated from different hot springs in Japan (Uchino & Doi 1967). Alicyclobacillus species are also found in soil which acts as the main reservoir of these bacteria (Goto et al., 2008;Groenewald et al., 2008;Wang et al., 2010). In addition, the soil is an important source of fruit contamination by Alicyclobacillus spores, which are transmitted by wind or during the harvesting period. For several years, the fruit juice industry has been affected by the problem of deterioration of fruits by Alicyclobacillus species (Sokołowska et al., 2020).
The metabolic products of these bacteria, which include halophenols (2,6-dibromophenol and 2,6-dichlorophenol) and guaiacol, are responsible for the formation of unusual medical, phenolic off-flavor and for the adverse changes noted in the taste and aroma of the fruit juice products (Pettipher et al., 1997;Orr et al., 2000). Furthermore, Alicyclobacillus spores are heat-resistant and can therefore survive pasteurization. The thermal and acid resistance of Alicyclobacillus is the result of the unique composition of fatty acids in the cell membrane of these organisms. These fatty acids contribute to increased packing of lipids in the membrane core and the stabilization of the membrane structure of the bacteria, which in turn allow them to tolerate high temperatures and highly acidic conditions (Kannenberg et al., 1984;Chang et al., 2004;Karavaiko et al., 2005;Ciuffreda et al., 2015).
16S rDNA gene is an essential tool in taxonomy, especially considering the large and still-growing database of 16S rDNA sequences, derived from diverse microorganisms. However, it does not always allow distinguishing closely related species; in addition, most microorganisms have several non-identical copies of this gene in their genome, which makes PCR-RFLP analysis difficult. Thus, we decided to include the rpoB gene in our taxonomy studies. RpoB encodes the β-subunit of bacterial RNA polymerase. This is a housekeeping gene that occurs in the genome in one copy, is widely used in taxonomy, and has already been used to identify and differentiate Alicyclobacillus strains (Dekowska et al., 2018).
To date, there have been no reports on the guaiacolproducing ability of A. fastidiosus. In A. acidoterrestris, guaiacol production is determined by the vdc operon consisting of three genes: vdcB, vdcC, and vdcD (Matsubara et al., 2003). The presence of this operon was confirmed in A. acidoterrestris, A. acidophilus, and A. herbarius. The operon sequence has been proven to be a useful marker in differentiating Alicyclobacillus strains that are capable of producing guaiacol (Dekowska et al., 2018).
In this study, we aimed to confirm the presence of Alicyclobacillus bacteria in Polish fruit orchards and characterize the three atypical A. fastidiosus strains that were isolated for the first time from the orchards in the Mazovia region of Poland.

Bacterial strains
The bacteria tested in the study were isolated from the samples taken from the top layer of soil and from the apple and pear fruits that were hand-collected from various orchards in the Mazovia region of Poland.

Alicyclobacillus species isolation and differentiation from other thermo-acidophilic bacterial species
The taint-producing Alicyclobacillus species was isolated using the International Federation of Fruit Juice Producers (IFU) Method No. 12 (IFU Method No. 12, 2004. Twenty soil samples and 20 fruit samples were taken manually from the five orchards in the Mazovia region for the study. The fruit or soil samples (10 g) were mixed with 90 ml of BAT broth (Merck, Darmstadt, Germany) and were heat-treated at 80°C for 10 min. This high-temperature treatment eliminated other vegetative microbial contaminants and induced the germination of spores. Following the heat treatment, the samples were incubated for 5 days at 45°C. Then, a loop of suspension was transferred onto BAT agar (pH 4.0±0.1) (Merck), and the plates were incubated for 3 days at 45°C. The cell morphology of all the isolated strains was studied by Gram staining and observation under an Olympus CX40 microscope (oil immersion, ×1500 magnification). Then, single colonies were spread-plated onto Plate Count Agar (PCA, pH 7.0±0.2) (Merck) to exclude the presence of Bacillus. In addition, the colonies assumed to be belonging to the genus Alicyclobacillus were spread-plated onto BAT agar and incubated at 65°C for 3 days. The presumptive strains were confirmed as taint-producing Alicyclobacillus if they could not grow on the BAT agar medium at a temperature of 65°C IFU Method No. 12, 2004.
According to the IFU Method No. 12, the isolated strains that potentially belonged to the Alicyclobacillus family were tested for the ability to produce guaiacol at 45°C (Niwa & Kuriyama, 2013). A 24-h plate culture of Alicyclobacillus species capable of causing spoilage was added to 1 ml BAT broth with vanillic acid (100 mg/l) and incubated for 3-4 h at 45°C. After incubation, guaiacol was detected through its oxidation by peroxidase (50 µl of 0.01% peroxidase in 500 µl of phosphate buffer (pH 6.0±0.1)), in the presence of hydrogen peroxide (50 µl of 0.5% H 2 O 2 ). The reaction resulted in the formation of brown-colored 3,3′-dimethoxy-4,4′-biphenoquinolone.
The growth of A. acidoterrestris was confirmed if the bacterial colony showed a green color, accompanied by a change in the color of the medium from blue to yellow (green color was observed during incubation at 45°C). Furthermore, a lack of growth was observed at 65°C for this species. Other Alicyclobacillus species (e.g. A. acidocaldarius) showed blue colonies on erythritol agar due to the lack of acid formation from erythritol at both 45°C and 65°C.

Identification of bacterial isolates via 16S rDNA gene sequence analysis
The bacterial isolates that were assumed to be belonging to the Alicyclobacillus genus were subjected to 16S rDNA gene sequence analysis. DNA was isolated from the bacterial cells using Genomic DNA Purification Kit (EUR X , Gdańsk, Poland) according to the manufacturer's instruction. The fragment of the 16S rDNA gene (1500 bp) was amplified and sequenced using primers fD1 (5′-AGAGTTTGATCCTGGCTCAG) and Alicyc-16srev (5′-ACGGCTACCTTGTTACGACT). The composition of the PCR mixture was as follows: 5 ng of template DNA, 50 pM of each of the primers, 25 µl of DreamTaq™, and Green PCR Master Mix (Thermo Scientific, Wallham, USA) with water added to obtain a final volume of 50 µl. PCR was performed under the following conditions: initial denaturation at 94°C for 2 min, followed by 40 cycles of denaturation at 94°C for 30 s, annealing at 51°C for 35 s, elongation at 72°C for 1 min and 40 s, and the final elongation at 72°C for 2 min. The obtained sequences were assembled using Serial Cloner (Serial Basic) and compared to the GenBank database using BLAST. DNA sequences obtained during this study were deposited in GenBank, and their accession numbers are given in Table 1.

RFLP analysis of rpoB gene fragment and vdc operon fragment
The RFLP analysis was carried out as described by Dekowska's group (Dekowska et al., 2018). Briefly, the rpoB gene fragment (1735 bp) was amplified using Gru3-Gru6 primers, the sequences of which were as follows: Gru3: 5′-CGYGACGTDCACTAYTCBCACTA, Gru4: 5′-GCCCANACYTCCATCTCRCCRAA, Gru5: 5′-CGCGACGTACACTATTCGCACTA, and Gru6: 5′-GCCCAAACCTCCATCTCACCAAA. The primers Gru5 and Gru6 were nondegenerated versions of the primers Gru3 and Gru4, respectively. The nondegenerated primers were used for amplifying the A. acidoterrestris samples, while the degenerated ones were used for amplifying other samples. All the primers were designed based on the comparison of rpoB sequences isolated from several Alicyclobacillus species, Bacillus subtilis, and Geobacillus stearothermophilus published in GenBank.
The composition of the PCR mixture was as follows: 30 ng of template DNA, 40 pM of each of the primers, 25 µl of DreamTaq™, and Green PCR Master Mix (Thermo Scientific) with water added to obtain the final volume of 50 µl. PCR was performed under the following conditions: initial denaturation at 94°C for 2 min, followed by 40 cycles of denaturation at 94°C for 30 s, annealing at 57°C (for the Gru5 and Gru6 primers) or at 59°C (for the Gru3 and Gru4 primers) for 35 s, elongation at 72°C for 1 min 45 s, and the final elongation at 72°C for 2 min.
The PCR mixture contained 25 ng of template DNA, 15 pM of each of the primers, 25 µl of DreamTaq™, and Green PCR Master Mix (Thermo Scientific) with water added to obtain the final volume of 50 µl. PCR was performed under the following conditions: initial denaturation at 94°C for 2 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 56°C for 35 s, elongation at 72°C for 1 min 50 s, and the final elongation at 72°C for 2 min.
The PCR-RFLP assay was performed according to the following procedure. First, 8-12 µl of the PCR products was digested with endonuclease HphI (Thermo Scientif-ic) in a volume of 20 µl. Following incubation at 37°C for 1-2 h and inactivation of the enzyme (10 min, 80°C), the samples were analyzed on a 2.5-3.0% agarose gel.
To determine the growth pH range of A. fastidiosus, the pH of the BAT broth was adjusted by adding 1 M sulfuric acid to achieve the following values: 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, and 5.5. The medium was inoculated with 18-h bacterial culture and incubated at an optimal temperature of 40°C for 24 h, 48 h, and 72 h. Following each incubation period, the optical density of the bacterial suspension was measured.

Assessment of the guaiacol-producing ability of A. fastidiosus isolates at three different temperatures
The ability to produce guaiacol was tested using the peroxidase method (Niwa & Kuriyama, 2013). The f1, f2, and f3 strains of A. fastidiosus were cultivated on BAT agar plates for 72 h at three different temperatures: 20°C, 25°C, and 45°C. Briefly, the BAT broth medium supplemented with vanillic acid (100 mg/l) was inoculated with a loop of harvested cells of A. fastidiosus. The medium was incubated for 7 h at 20°C, 25°C, and 45°C. If the guaiacol was present, the color change of the medium containing vanillic acid and the bacterial strain was observed after adding an appropriate amount of phosphate buffer, peroxidase, and hydrogen peroxide.

Identification of Alicyclobacillus bacteria based on culture-dependent methods
The samples of the top layer of soil as well as the apple and pear fruits collected from the orchards in the Mazovia region, one of the biggest fruit-producing regions of Poland, were screened for the presence of taintproducing Alicyclobacillus. Eight isolated bacterial strains were characterized as Gram-positive, spore-forming bacilli and were found to be able to grow on BAT agar at 45°C, whereas at the same temperature they could not grow on PCA. Based on these observations, they were classified as the members of Alicyclobacillus genus. Furthermore, these isolates could not grow at 65°C, which is a characteristic of presumptive taint-producing Alicyclobacillus species (IFU Method No. 12, 2004. All the eight isolated strains exhibited the ability to produce guaiacol at 45°C. According to Baumgart and others (Baumgart et al., 2003), the species A. acidoterrestris has the ability to utilize erythritol as a carbon source for acid formation, which is indicated by the growth of yellow-green colonies on erythritol agar with bromophenol blue. In the present study, five acidophilic strains -strain 1, strain 2, strain 3, strain 4, and strain 5 -isolated from apples and soil samples collected from apple orchard were able to grow on erythritol agar and formed yellow-green colonies, whereas three acidophilic strains -strain f1, strain f2, and strain f3 -isolated from pears and soil samples collected from pear orchard formed blue colonies on this medium. Taking these results into account, we can assume that strains 1-5 belong to the species A. acidoterrestris, whereas strains f1, f2, and f3 do not belong to this species although they showed guaiacol-producing ability.

Sequence analysis and PCR-RFLP
The 16S rDNA sequence analysis indicated that strains f1, f2, and f3 probably belong to the species A. fastidiosus. The 16S rDNA sequences of these isolates showed the highest identity (>99%) to those of the strain A. fastidiosus DSM 17978 (GenBank accession number AB264021) and two other A. fastidiosus strains (NR_041471 and NR_114208). On the other hand, strains 1-5 were identified as A. acidoterrestris (>99% identity to A. acidoterrestris).
RFLP analysis was performed for the rpoB gene fragment of all the analyzed Alicyclobacillus strains, and their patterns were compared to those of the following reference strains: A. acidoterrestris DSM 2498, A. acidoterrestris ATCC 49025, A. acidiphilus DSM 14558, A. herbarius DSM 13609, A. hesperidum DSM 12489, A. acidocaldarius DSM 446 (Fig. 1a), and A. fastidiosus DSM 17978 (Fig. 1b). The RFLP patterns of strains f1 and f3 were identical to that of A. fastidiosus DSM 17978, while the pattern of strain f2 was not identical but similar to that of the mentioned strain. Based on the comparison of the RFLP patterns of all A. acidoterrestris strains and isolates f1, f2, and f3, we excluded the possibility that strains f1, f2, and f3 belong to A. acidoterrestris species, whereas the rest of the analyzed strains were found to show identical patterns as A. acidoterrestris type II ATCC 49025 ( Fig. 1a  and 1b). So far, two types (type I and type II) of A. acidoterrestris strains have been reported (Durak et al., 2010, Osopale et al., 2016, Dekowska et al., 2018. In this study, we observed only type II A. acidoterrestris. Dekowska's team (Dekowska et al., 2018) analyzed 60 A. acidoterrestris species isolated from Polish fruits and fruit products and showed that the distribution of these species was quite even with 27 belonging to type I and 33 to type II.

a b
The performed PCR assay targeting the vdc operon fragment resulted in a product of about 1800 bp for all the tested strains, regardless of whether they were classified as A. acidoterrestris or A. fastidiosus. A band of identical size was obtained for both reference strains of A. acidoterrestris, but not for the reference strain A. fastidiosus DSM 17987. The strain A. fastidiosus DSM 17987 probably carries a defective version of the operon as Wang and coworkers (Wang et al., 2021) obtained a PCR product for this strain using primers designed for vdcC gene, whereas they did not observe guaiacol production. This suggests that whole-genome sequencing or further study on this particular region is required to shed more light on this issue.
The RFLP analysis of the vdc operon fragment showed that the patterns obtained for the environmental samples classified as A. acidoterrestris were identical to those of A. acidoterrestris type II ATCC 49025, while the patterns obtained for the environmental samples classified as A. fastidiosus were identical to each other and showed no similarity to the patterns of any reference strain. The strain A. fastidiosus DSM 17978 produced no band in the PCR assay and, therefore, it was excluded from the RFLP analysis (Fig. 2).
Sequences of the analyzed vdc fragment (GenBank accession number MH938036) were found to be identical to those of all three strains of A. fastidiosus isolated in this study, and they showed 80%, 76%, and 71% identity to the sequences of A. acidoterrestris ATCC 49025, A. acidiphilus NBRC 100859, and A. herbarius DSM 13609, respectively (BLAST search against nucleotide and WGS database). The BLAST search revealed no sequence with more than 80% identity to that of the analyzed samples.

Morphological and physiological characterization of A. fastidiosus
Strains f1, f2, and f3 stained Gram-positive and produced flat, creamy-white colonies. When viewed under the microscope, they appeared as rods of a length of 4.0-4.5 μm and a width of 0.8-1.0 μm. The growth temperature range of all these strains was found to be 20-55°C with optimal growth noted at 30-45°C. All three strains of A. fastidiosus grew in the pH range of 3.0-5.0 with an optimum of 3.0-3.5. These results differ from the optimum values reported by Goto's group  because in their research the optimum pH was 4.0-4.5 and the optimum temperature was 40-45°C.
Furthermore, the examined strains f1, f2, and f3 exhibited a common characteristic: they were able to produce guaiacol from vanillic acid at a temperature of 20°C, 25°C, and 45°C and at an optimal pH of 3.0-3.5. Another important biochemical feature of these strains was the lack of acid formation from erythritol. This was evidenced by the absence of a color change of the medium containing erythritol and bromophenol blue. However, all the isolated strains of A. acidoterrestris were able to use erythritol as a carbon source for the formation of acids. The phenotypic characteristics of the isolated A. fastidiosus strains and their comparison with the characteristics reported by Goto and coworkers (Goto et al., 2007) are shown in Table 2.
The classification of strains f1, f2, and f3 as A. fastidiosus was strongly supported by DNA sequencing analy-   sis, RFLP assay, and morphological characterization. The geographical and ecological distribution is considered an important source of information on spoilage species for the beverage industry. As far as we know, this study is the first to report the isolation of A. fastidiosus species from Polish pear orchard, and the second to report the isolation of these bacteria in the world. Strains f1, f2, and f3 were mostly characterized by similar morphological and biochemical properties as A. fastidiosus species described by Goto and others . According to these authors, A. fastidiosus species does not exhibit the ability to produce guaiacol. These results differ from those observed in the guaiacol production test carried out in the present study. The test showed that all the environmental strains of A. fastidiosus and A. acidoterrestris, as well as the reference strains of A. acidoterrestris, were able to produce guaiacol from vanillic acid at 45°C. Unlike the other spoilage bacteria belonging to the Alicyclobacillus genus, A. fastidiosus was proven to produce guaiacol after 7 h of incubation at a temperature of 20°C, which may pose a serious threat to the fruit juice industry. Thus far, only A. acidoterrestris species has been identified as capable to produce guaiacol at 21-25°C, which is an unfavorable temperature for the growth of most thermophilic species of Alicyclobacillus (Pettipher et al., 1997, Orr et al., 2000. According to Orr and coworkers (Orr et al., 2000), guaiacol production by A. acidoterrestris can be noticed after 8 days of incubation in juice at 21°C. By contrast, Sokołowska's group (Sokołowska et al., 2013) reported that A. acidoterrestris strains were able to produce guaiacol only after 34 days of incubation in juice at a temperature of 25°C. The species A. fastidiosus is closely related to A. acidoterrestris, which is also a guaiacol-producing bacterium, most commonly isolated from fruit raw materials and fruit products.
In conclusion, we suggest that A. fastidiosus should be added to the group of spoilage species.