Effects of Temperature on Resistance to Streptomycin in Xanthomonas arboricola pv. pruni

Bacterial distribution and culture

To confirm antibiotic resistance, Xanthomonas arboricola pv. pruni (Xap) strain KACC 19949 was obtained from the Korean Agricultural Culture Collection (KACC) of the National Institute of Agricultural Science. The strain was stored as a stock culture at −80°C.

Minimum inhibitory concentration analysis of Xap in response to streptomycin treatment

To measure the minimum inhibitory concentration (MIC) of streptomycin against Xap, streptomycin was added to the LB medium (Difco, Becton Dickinson, Sparks, MD, USA) at different final concentrations (0.25, 0.5, 1, 2, 4, 10, 20, 30, 40, 50, 100, and 1,000 μg/ml). Xap strains were seed-cultured in 5 ml of LB medium (Luria Bertani medium, Difco, Becton Dickinson) at 28°C for 24 h. Then, 100 μl of seed culture was used to inoculate 1 ml of LB medium containing streptomycin at a certain concentration in a 15-ml Falcon tube and cultured for 24 h at 28°C, with shaking at 120 rpm. Bacterial growth was evaluated by measuring the optical density at 600 nm using a Synergy HT MultiMode Microplate Reader (BioTek Instruments, Winooski, VT, USA), and growth was considered to have occurred if the absorbance was 0.1 or higher (Lyu et al., 2019). Subsequently, the culture was diluted to 10⁻⁶ to determine the viable cell count, and the colony count was measured after spreading the culture on LB agar plates.

Changes in streptomycin resistance due to temperature

The streptomycin resistance of Xap at low temperatures was tested through culturing at 10°C; however, since bacterial growth was insufficient, the bacteria were incubated in antibiotic media at each temperature for 24 h instead of culturing, and then bacterial growth was compared by culturing at 28°C. After adjusting the OD₆₀₀ to 1.0 using the seed culture of Xap in LB medium for 24 h, 100 μl of the seed culture was added to 15-ml Falcon tubes containing 1 ml of LB medium containing streptomycin at a certain concentration. After culturing at 10°C, 25°C, and 30°C for 24 h with shaking at 125 rpm, the liquid medium at each concentration was transferred to a 1.5-ml tube and centrifuged at 13,000 rpm for 3 min.

After removing the supernatant, 1 ml of sterilized water was added back into the 1.5-ml tube, vortexed, and centrifuged, leaving behind only the pellet. This process was repeated twice. Afterwards, the washed pellet was suspended in 1 ml of LB medium. Then, 100 μl of each the suspension was added to a test tube containing 900 μl of LB medium and incubated, with shaking, at 28°C for 24 h, and the absorbance was measured; absorbance > 0.1 was considered to indicate growth.

Through the newly established process, the bacteria that grew depending on the temperature in each antibiotic were washed to remove the antibiotics present in the medium and replaced with LB medium, thereby clearly confirming the growth of the bacteria. The samples were serially diluted to 10⁻⁶, plated on LB agar, and the colonies were enumerated to determine cfu.

Changes in occurrence frequency of streptomycin-resistant bacteria according to temperature

To determine the occurrence frequency of antibiotic resistance at 28°C, 100 μl of LB medium was added to a 96-well plate with streptomycin concentrations of 10, 25, and 50 μg/ml. Then, 10 μl of the Xap suspension (OD₆₀₀ = 0.1), prepared by culturing the strain in LB medium for 24 h, was inoculated and cultured in a shaking incubator at 120 rpm for 3 days. Absorbance was measured at 600 nm. Each well with a value of 0.1 or higher was inoculated into LB agar medium containing 100 μg/ml streptomycin by dipping a sterilized toothpick into the medium. To check the frequency of resistance caused by temperature, 10 μl of the LB-cultured suspension (OD₆₀₀ = 0.1) was inoculated into 100 μl of LB medium containing 25 μg/ml streptomycin in a 96-well plate, and incubated at 20°C, 25°C, 28°C, and 30°C following the same procedure as above.

Genetic mutation analysis of resistant bacteria

Primers listed in Table 1 were used to amplify and analyze the strA, strB, and rpsL genes of streptomycin-resistant strains. The strA gene was amplified using the strAP-F and strAT-R primers, and the strB gene was amplified with strBP-F and strBT-R primers. These primers were used as described in Herbert et al. (2022). PCR conditions were as follows: denaturation at 95°C for 5 min, followed by 34 repetitions of denaturation at 95°C for 15 s, annealing at 55°C for 20 s, and extension at 72°C for 90 s, and a final extension at 72°C for 5 min. The PCR amplification of the 16S rRNA gene was performed using universal primers, 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTT-ACGACTT-3′). The PCR conditions consisted of initial denaturation at 96°C for 4 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 57°C for 30 s, and extension at 72°C for 1 min, and a final extension at 72°C for 10 min.

Table 1

Primers used in this study

To amplify the rpsL gene, specific primers (rpsL-F and rpsL-R) were designed using the Primer 3 online program (https://primer3.ut.ee/) based the Xap genome information in the NCBI database, and the specificity of the primers was confirmed using BLAST. The PCR conditions included an initial denaturation at 95°C for 5 min, followed by 30 cycles of denaturation at 95°C for 60 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s, and a final extension at 72°C for 5 min. Following the final extension, the reaction was held at 4°C for storage. The PCR reaction solution consisted of 2 μl of 10× reaction buffer, 0.4 μl of 10 mM dNTP, 1 μl of each primer (10 mM), 0.1 μl of 5.0 U of Tap DNA polymerase (BioFact Inc., Daejeon, Korea), and 2 μl of genomic DNA template (50 ng/μl). The amplified gene fragment was verified using 1.5% agarose gel electrophoresis in 0.5× TBE buffer, followed by bidirectional sequencing analysis performed by Macrogen Inc. (Seoul, Korea).

Statistical analysis

The experimental results were analyzed using the R software. Differences in the occurrence frequency of resistant strains were assessed through ANOVA, followed by Tukey’s HSD post hoc test, and significant differences between treatment groups were identified at P < 0.05. The results were grouped and presented in a bar plot.

Measurement of MIC of Xap against streptomycin at incubation temperature

To measure the MIC of Xap against streptomycin at 28°C, the degree of bacterial growth in streptomycin (0.25 to 1,000 μg/ml) was determined by measuring absorbance. In the case of medium without antibiotics, the absorbance was 0.7 ± 0.04 at OD600 after 24 h, and it was confirmed that the absorbance was maintained at 0.5 at OD₆₀₀ in concentrations up to 10 μg/ml of streptomycin (Fig. 1A).

Fig. 1

Measurement of the minimum inhibitory concentration (MIC) of Xanthomonas arboricola pv. pruni against streptomycin at 28°C. (A) MIC values measured at streptomycin concentrations ranging from 0.25 to 1,000 μg/ml. (B) MIC values measured at streptomycin concentrations ranging from 10 to 20 μg/ml. The negative control is labeled as Negative, which is a treatment group that was not inoculated with bacteria. (C) Viable cell counts were assessed at various streptomycin concentrations ranging from 0.25 to 1,000 μg/ml. The data are expressed as Log₁₀ (cfu/ml) and represent the mean ± standard deviation of three independent replicates.

On the other hand, it was confirmed that bacteria could not be cultured at a concentration of 20 μg/ml or higher (Fig. 1A). In order to measure the MIC more precisely, the concentration was increased by 2 μg/ml from 10 μg/ml to 20 μg/ml to check the degree of growth. As a result, at 18 μg/ml, OD₆₀₀ decreased to 0.25 ± 0.009, and it was confirmed that bacteria were not cultured at 20 μg/ml (Fig. 1B). Therefore, the MIC of Xap streptomycin at 28°C was confirmed to be 20 μg/ml. The MIC was measured using the dilution plate method to check the number of viable bacteria according to the antibiotic concentration. As a result, the number of viable bacteria decreased by half (to a total of 10⁴ cfu/ml) from 10 μg/ml to 20 μg/ml, and it was confirmed that bacteria were not cultured at 30 μg/ml (Fig. 1C).

Analysis of change in the response of Xap to streptomycin due to temperature

To test the change in the MIC of Xap against streptomycin due to temperature, bacterial growth was measured at concentrations ranging from 5 to 25 μg/ml. In order to analyze the response of antibiotics to changes in temperature, a new method for testing the changes in antibiotics according to temperature was established (Fig. 2A). As a result of confirming the change in antibiotic response due to temperature through the obtained method, the culture concentration of bacteria was found to be up to 10 μg/ml at 10°C, which was not statistically different from the medium without antibiotics. On the other hand, at a concentration of 15 μg/ml, an OD₆₀₀ value of 0.2 was observed, indicating approximately half the growth, while no growth was confirmed at 20 μg/ml, indicating that culture did not occur (Fig. 2B).

Fig. 2

Results of minimum inhibitory concentration (MIC) measurements for streptomycin at 25 μg/ml at different temperatures. (A) MIC measurement method, with an added washing step at various temperatures. (B) MIC measured at 10°C. (C) MIC measured at 25°C. (D) MIC measured at 30°C. MIC measured at 10°C (E), 25°C (F), and 30°C (G), at streptomycin concentrations of negative, 5, 10, 15, and 20 μg/ml. Viable cell counts were expressed as Log₁₀(cfu/ml) and are shown as mean ± standard deviation of three independent biological replicates. The negative control is labeled as negative, which is a treatment group that was not inoculated with bacteria. LB, Luria-Bertani.

At 25°C, the medium without antibiotics had a culture concentration with an OD₆₀₀ of 0.7 ± 0.02, while the medium containing 10 μg/ml antibiotics showed an OD₆₀₀ value of 0.7 ± 0.02, which was indicative of statistically significant inhibition of bacterial growth (Fig. 2C). On the other hand, at 30°C, the OD₆₀₀ value of the medium without antibiotics was 0.8 ± 0.03, and it was confirmed that OD₆₀₀ value decreased sharply to 0.1 at a concentration of 10 μg/ml (Fig. 2D). At 15 μg/ml, it was confirmed that the OD₆₀₀ value was the same as that of the untreated bacteria at both 25°C and 30°C (Fig. 2). When examining the response to antibiotic concentrations at different temperatures, it was observed that a concentration of 20 μg/ml did not support any growth at any of the tested temperatures. At a concentration of 10 μg/ml, the 10°C treatment group showed no statistically significant difference compared with the medium without antibiotics. However, the resistance of Xap to streptomycin was confirmed to increase at 30°C compared with 10°C and 25°C (Fig. 2B–D). In addition, when the viable cell count was measured using the dilution plate method, the viable cell count decreased statistically significantly from 15 μg/ml at all concentrations. Specifically, a decrease in the viable cell count was observed at 10 μg/ml at 30°C compared with 10°C and 20°C (Fig. 2E–G). These results suggest that the response of bacteria to antibiotics may vary depending the temperature.

Analysis of change in the frequency of Xap resistance to streptomycin caused by temperature

As a result of the above, it was possible to verify the change in the MIC of Xap in relation to streptomycin concentration at different temperatures. Additionally, a 96-well plate system was constructed to study not only the change in MIC but also the frequency of occurrence of resistant colonies (Fig. 3A). After inoculating the Xap cultures into each 96-well plate, the occurrence of resistant bacteria was observed at each concentration, and resistance was further validated on a solid medium containing antibiotics at a concentration of 100 μg/ml (Fig. 3A).

Fig. 3

Measurement of antibiotic resistance frequency at different concentrations. (A) Measurements were taken in a 96-well plate at 28°C with streptomycin concentrations of 10, 25, and 50 μg/ml. Wells that showed bacterial growth only in the liquid medium were classified as non-resistant, while those that exhibited growth in both the liquid wells and LB agar medium containing 100 μg/ml of streptomycin were classified as resistant. NC is negative control. (B) The proportion of resistance observed in the 96-well plate. (C) The proportion of growth observed in the resistance medium. (D) The ratio of non-growth to resistant.

For each streptomycin concentration, growth was observed in six replicates using a 96-well system, resulting in a total of 876 wells. As a result, 692 wells were used for culturing at 10 μg/ml, 257 wells at 25 μg/ml, and 319 wells at 50 μg/ml (Fig. 3B). The culture was collected from each well and plated on LB solid medium containing 100 μg/ml streptomycin to assess the presence or absence of resistance. On the solid medium, resistant bacteria were confirmed to grow in 8 wells out of 507 for 10 μg/ml, 7 wells out of 205 for 25 μg/ml, and 7 wells out of 92 for 50 μg/ml (Fig. 3C). Therefore, the exact rate of resistant bacterial occurrence was determined by dividing the number of wells with resistant colonies by the total number of wells with cultured bacteria. The rate of resistant bacterial occurrence was calculated as 1.6% at 10 μg/ml, 3.4% at 25 μg/ml, and 7.6% at 50 μg/ml, indicating an increase in the occurrence rate with rising streptomycin concentration (Fig. 3D).

The occurrence frequency of resistance according to temperature was tested in seven replicates (Fig. 4A). The ratio of wells cultured to wells containing 25 μg/ml streptomycin was 0.48% (6 wells out of 1224) at 20°C, 4.5% (40 out of 780) at 25°C, 6.8% (75 out of 966) at 28°C, and 6.2% (45 out of 750) at 30°C (Fig. 4B). When the culture fluid was collected from the wells and dispensed on solid medium containing 100 μg/ml streptomycin, fluid from zero wells showed growth at 20°C, 2 wells at 25°C, 8 wells at 28°C, and 15 wells at 30°C (Fig. 4C). The number of wells cultured in the primary culture medium and the occurrence rate of resistant strains on the secondary solid medium were the highest (6.5% at 25°C, 10.7% at 28°C, and 33.3% at 30°C). On the other hand, no resistant strains appeared in the secondary culture at 20°C (Fig. 4D).

Fig. 4

Measurement of antibiotic resistance frequency at different temperatures. (A) Measurements were taken in a 96-well plate containing 25 μg/ml streptomycin at temperatures of 20°C, 25°C, 28°C, and 30°C. Wells that only showed growth were classified as non-growth, while those that grew in both the wells and Luria-Bertani (LB) agar medium containing 100 μg/ml of streptomycin were classified as resistant. (B) The proportion of resistance observed in the 96-well plate. (C) The proportion of growth observed in the resistance medium. (D) The ratio of non-growth to resistant.

Genetic mutation assay of resistant colonies

In order to identify genetic mutations in the resistant colonies cultured in the second experiment, the analysis focused on insertions in the strA and strB genes, known as existing resistance mechanisms, and point mutations in the rpsL gene. After selecting resistant colonies that had undergone secondary culture at each temperature, DNA was extracted, and PCR was performed using each specific primer. As a result, in the case of positive control, the strA and strB genes were detected, whereas in the isolated resistant strain, the strA and strB genes were not observed (Fig. 5A).

Fig. 5

Amplification of the strA, strB, and rpsL genes in streptomycin-resistant strains of Xanthomonas arboricola pv. pruni. (A) Agarose gel electrophoresis of PCR products amplified using the strA/strB primers, strAP-F and strAT-R, strBP-F and strBT-R, and 16S-F and 16S-R primers. Lane X represents Xanthomonas arboricola pv. pruni. Lane M is a 100 bp ladder; lanes 1–5 show no-growth strains: G9 (lane 1), B9 (lane 2), C10 (lane 3), E8 (lane 4), and B3 (lane 5); lanes 6–9 show resistant strains: C6 (lane 6), B7 (lane 7), D6 (lane 8), and F11 (lane 9). (B) Sequencing results obtained using the rpsL-F and rpsL-R primers. S is a susceptible strain; XAP₁ refers to Xanthomonas arboricola pv. pruni KACC 19949; and XAP₂ refers to Xanthomonas arboricola pv. pruni KACC 19949, and XAP² referring to Xanthomonas arboricola pv. pruni strain F1 107. ‘SR’ refers to wells that showed no growth in the 96-well plate (non-growth), while RR indicates resistant wells that showed growth in both the 96-well plate and resistance medium. The * symbol indicates a mutated nucleotide sequence.

In the case of point mutation occurrence in the rpsL gene, point mutations in the rpsL gene were not confirmed in the streptomycin-sensitive strain (S) and in the strain cultured only in the primary well (SR) (Fig. 5B). On the other hand, in the strain cultured on secondary solid medium, it was revealed that alanine (A) was converted to glycine (G) in codon 88 (Fig. 5B).