3.1 XOS consumption by LAB strains derived from rabbits
Thirty-four LAB strains (36.2%, 34/94) possessed the capability of utilizing XOS, including P. acidilactici (n = 3), L. plantarum (n = 3), W. kandleri (n = 14), L. brevis (n = 7), and Lactobacillus sp. (n = 7) (Table S1), and the proportion of XOS-utilizing LAB strains belonging to these five species was shown in Table 1. All of LAB strains belonging to species including W. kandleri, L. brevis and Lactobacillus sp. were capable of utilizing XOS, indicating that these three species from the rabbits’ gut possessed the potential to proliferate and further benefit host health through utilizing XOS.
Table 1
XOS consumption by LAB strains derived from rabbits
Species
|
Total number
|
XOS-utilizing strainsa
|
Ratiob
|
Weissella kandleri
|
14
|
14
|
100%
|
Levilactobacillus brevis
|
7
|
7
|
100%
|
Lactobacillus sp.
|
7
|
7
|
100%
|
Lactiplantibacillus plantarum
|
32
|
3
|
9.4%
|
Pediococcus acidilactici
|
14
|
3
|
21.4%
|
Lacticaseibacillus paracasei
|
7
|
0
|
0
|
Latilactobacillus curvatus
|
6
|
0
|
0
|
Weissella paramesenteroides
|
5
|
0
|
0
|
Weissella cibaria
|
2
|
0
|
0
|
In total
|
94
|
34
|
36.2%
|
a The capability of XOS consumption by LAB strains derived from rabbits was evaluated through fermentation tests using bMRS media containing 1% (w/v) xylo-oligosaccharides (XOS) as a carbon source. A XOS-utilizing strain was identified to be a responder by a color change from purple to yellow in the media as indicated by bromocresol purple. |
b The data exhibited the proportions of XOS-utilizing strains in each species or in total LAB strains. |
The genus Weissella are obligate heterofermentative LAB, primarily producing lactic acid through fermenting carbohydrates. This genus has been found to have great potential for use in pharmaceutical, medical, and food industries due to their capabilities of producing exopolysaccharides and bacteriocins. However, the capability of prebiotics oligosaccharides consumption by the Weissella strains was little known[33–35]. In the present study, all W. kandleri strains could metabolize XOS, and their growth on media containing XOS as a sole carbon source is similar to that on glucose. In contrast, W. cibaria strains derived from rabbits’ gut were not capable of utilizing XOS. Up to date, a few L. brevis strains such as L. brevis DSMZ 1264, L. brevis DSMZ 1269, and L. brevis NRRL 1834 have been reported to be able to utilize XOS. Similarly, all L. brevis strains derived from rabbits in the present study were also capable of utilizing XOS; however, these L. brevis strains showed significantly different XOS-utilizing capabilities, some of which grew better on XOS than on glucose [36–39], suggesting a significant strain-specificity within L. brevis species in terms of XOS consumption. In addition, only three L. plantarum strains showed XOS-utilizing capability, while other twenty-nine L. plantarum strains could not grow on media containing XOS as a sole carbon source. A previous study also evidenced that several L. plantarum strains derived from traditional fermented foods was able to achieve growth through utilizing XOS as an effective substrate[40].
Pediococcus acidilactici is one kind of probiotic that is permitted to be used as a feed additive in many countries[20]. In a previous study, it was also evidenced that there was a prebiotic activity of XOS on P. acidilactici and the efficacy was significantly better than inulin, fructooligosaccharide (FOS) and isomaltooligosaccharide (IMO)[41]. In addition, XOS also could significantly promote the proliferation of P. acidilactici BCC-1, which contributes to enhance gut health and growth performance of broilers[42]. P. acidilactici YT088 and YT089 in the present study showed a preference to utilize XOS. Therefore, we speculated that a combination of P. acidilactici YT088 (or YT089) and XOS in the form of a synbiotic might contribute to improve the gut health and growth performance of rabbits. In addition, the present study showed all Lactobacillus sp. strains derived from rabbits were capable of utilizing XOS efficiently; up to date, the prebiotic activity of XOS on this species and the underlying molecular mechanism were almost unknown. Therefore, Lactobacillus sp. YT155 was selected as a representative strain for further research.
3.2 Growth profile of XOS-utilizing LAB strains from rabbits’ gut
The growth profiles of XOS-utilizing LAB strains were investigated through determining OD600 nm and pH values of bacterial cultures after 72 h in bMRS broth containing 1% (w/v) XOS with the growth on glucose as a positive control. The results showed that the OD600 nm and pH values of thirty-four XOS-utilizing LAB strains were within the range of around 0.5 to 1.9 and 4.18 to 6.08, respectively (Table 2). A majority of XOS-utilizing LAB strains (88%, 30/34) grew very well on XOS compared with the growth on glucose as a carbon source in terms of the OD600 nm values. Moreover, 94% (32/34) of XOS-utilizing strains could significantly lower the pH through producing a large amount of SCFAs with a final pH lower than 5. Given that caecum is the primary site of fermentation, these results in the present study suggests that XOS is a kind of promising prebiotic which might be able to significantly promote the proliferation of these LAB strains derived from rabbits and lower the pH of the caecum of rabbits. Up to date, there were already a large number of studies focusing on the prebiotic effects of XOS on pig and poultry[13, 14, 16], while the impact of XOS on the gut microbiota was largely unknown. The present study firstly assessed the capability of XOS consumption by LAB strains derived from neonate rabbits and identified responders and nonresponders of XOS in rabbits’ gut through in vitro fermentation tests, which is quite critical for the application of XOS as a prebiotic in modulating gut microbiota of neonate rabbits during the early life.
Table 2
Growth profile of XOS-utilizing LAB strains from rabbits’ gut
Strain
|
OD600
XOS Glucose
|
pH value
XOS Glucose
|
Species
|
YT088
|
1.784
|
1.773
|
4.69
|
4.40
|
Pediococcus acidilactici
|
YT089
|
1.755
|
1.806
|
4.69
|
4.25
|
Pediococcus acidilactici
|
YT097
|
0.519
|
2.163
|
6.03
|
4.24
|
Lactiplantibacillus plantarum
|
YT100
|
1.251
|
1.039
|
4.74
|
4.79
|
Weissella kandleri
|
YT101
|
1.264
|
1.051
|
4.88
|
4.84
|
Weissella kandleri
|
YT102
|
1.213
|
1.053
|
4.74
|
4.93
|
Weissella kandleri
|
YT103
|
1.247
|
1.069
|
4.86
|
5.02
|
Weissella kandleri
|
YT104
|
1.237
|
1.054
|
4.74
|
4.79
|
Weissella kandleri
|
YT105
|
1.147
|
0.924
|
4.51
|
4.80
|
Weissella kandleri
|
YT106
|
1.100
|
1.108
|
4.78
|
4.72
|
Weissella kandleri
|
YT107
|
1.152
|
0.867
|
4.64
|
4.79
|
Weissella kandleri
|
YT108
|
1.559
|
0.925
|
4.69
|
5.28
|
Levilactobacillus brevis
|
YT109
|
1.601
|
0.876
|
4.71
|
5.43
|
Levilactobacillus brevis
|
YT110
|
1.945
|
2.074
|
4.18
|
4.29
|
Lactiplantibacillus plantarum
|
YT115
|
1.541
|
1.638
|
4.71
|
4.39
|
Lactobacillus sp.
|
YT116
|
1.548
|
1.624
|
4.66
|
4.44
|
Lactobacillus sp.
|
YT124
|
1.778
|
1.779
|
4.62
|
4.52
|
Pediococcus acidilactici
|
YT134
|
1.726
|
1.865
|
4.63
|
4.25
|
Levilactobacillus brevis
|
YT135
|
1.380
|
1.352
|
4.63
|
4.84
|
Levilactobacillus brevis
|
YT136
|
1.775
|
1.965
|
4.70
|
4.25
|
Levilactobacillus brevis
|
YT137
|
1.488
|
0.663
|
4.73
|
5.73
|
Levilactobacillus brevis
|
YT138
|
1.597
|
0.645
|
4.66
|
5.94
|
Levilactobacillus brevis
|
YT148
|
0.551
|
2.171
|
6.08
|
4.21
|
Lactiplantibacillus plantarum
|
YT151
|
1.450
|
1.734
|
4.76
|
4.40
|
Lactobacillus sp.
|
YT152
|
1.625
|
1.710
|
4.44
|
4.37
|
Lactobacillus sp.
|
YT153
|
1.505
|
1.512
|
4.71
|
4.44
|
Lactobacillus sp.
|
YT154
|
1.604
|
1.628
|
4.46
|
4.47
|
Lactobacillus sp.
|
YT155
|
1.150
|
1.617
|
4.72
|
4.44
|
Lactobacillus sp.
|
YT161
|
0.935
|
0.954
|
4.81
|
4.83
|
Weissella kandleri
|
YT162
|
1.300
|
1.206
|
4.80
|
4.81
|
Weissella kandleri
|
YT163
|
1.105
|
0.945
|
4.83
|
4.73
|
Weissella kandleri
|
YT164
|
1.335
|
1.039
|
4.71
|
4.79
|
Weissella kandleri
|
YT178
|
1.211
|
1.123
|
4.76
|
4.77
|
Weissella kandleri
|
YT179
|
1.157
|
1.046
|
4.63
|
4.76
|
Weissella kandleri
|
Thirty-four XOS-utilizing LAB strains were inoculated into bMRS broth supplemented with 1% (w/v) XOS as a carbon source and 1% (w/v) glucose as a positive control respectively. The growth of these XOS-utilizing strains was investigated by the determination of OD600 nm and pH values of bacterial cultures after 72 h of incubation. |
3.3 XOS metabolic properties of Lactobacillus sp. YT155
The OD600 nm and pH values of Lactobacillus sp. YT155 were depicted in Fig. 1, which was cultured in bMRS broth supplemented with different carbon sources (XOS and glucose) and water (negative control group), respectively. Except for negative control group without adding carbon source, Lactobacillus sp. YT155 exhibited a significant increase of biomass on both glucose and XOS (Fig. 1A) with similar growth rates and consistent growth cycle including lag phase from 0 h to 4 h, exponential phase from 4 h to 24 h, and stable phase from 24 h to 72 h. However, the OD600nm values of bacterial cultures of Lactobacillus sp.YT155 on XOS were much lower compared with the growth on glucose during the 72-hour fermentation. The fermentation of both glucose and XOS by Lactobacillus sp. YT155 resulted in a significant decline of pH values with final values being 4.4 and 4.5, respectively, and a similar decreasing rate of pH values was observed with a rapid decline at 12 h (Fig. 1B). Therefore, both the pH change and biomass increasing with a final OD600 nm value being 1.5 through utilizing XOS as a carbon source suggested that Lactobacillus sp. YT155 could efficiently metabolize XOS.
Although Lactobacillus sp. YT155 could proliferate and produce a large amount of acid through utilizing XOS based on the results of both OD600 nm and pH change, it was not immediately clear how XOS was consumed by strain YT155. Therefore, the XOS-utilizing pattern of strain YT155 was investigated by analyzing the depletion of XOS from the growth medium using TLC which could efficiently separate different DP of components through using sugars including 1% glucose (monomer) and 1% XOS as controls. As shown in Fig. 1C, the spot intensity of the supernatants of the bacterial culture at 0 h was consistent with the control group (1% XOS), indicating that XOS in the culture was not utilized by strain YT155. However, the spot intensity of cultures’ supernatant at 12 h was lighter compared to that of the control group (1% XOS), suggesting that XOS with a degree of polymerization (DP) ranging from around 2 to 5 was consumed to some extent. Also, the spot intensity of fermentation cultures was seen to become increasingly lighter with time during fermentation. In addition, it was observed that only a minimal XOS (around DP 4) was left in the culture after 72 h and other XOS components of the cultures were completely consumed by strain YT155 based on the spot intensity on the TLC bands. Therefore, the TLC analysis revealed that XOS oligomers were utilized by strain YT155 simultaneously, regardless of their chain length, which was similar to the previous reports[43].
As a consequence, the results confirmed that Lactobacillus sp. YT155 possessed a good capability to metabolize XOS, indicating that there may be functional genes encoding the XOS-metabolizing enzymes in the genomic sequence of strain YT155. In addition, it could be speculated that Lactobacillus sp. YT155 might possess good adhesion and colonization ability in rabbit intestines due to this strain being isolated from rabbit, which was one of the crucial factors of survival in the gut of rabbits[44]. Therefore, Lactobacillus sp. YT155 might be combined with XOS in a form of a synbiotic to regulate the gut microbiota in rabbits, thereby promoting gut health of rabbits during their early life.
3.3 Probiotic properties in vitro of Lactobacillus sp. YT155
If Lactobacillus sp. YT155 could be employed as a feed additive in rabbit breeding, then it must survive in a variety of environmental conditions in the production process, including pH, temperature, and salinity, etc. Therefore, the probiotic properties of Lactobacillus sp. YT155 were evaluated in vitro, and the results were shown in Fig. 2. First of all, a high tolerance was observed when Lactobacillus sp. YT155 was exposed to pH 5.0, 6.0, 8.0, 9.0, and 10.0, with a survival rate of 63.71% ± 4.45%, 92.64% ± 3.86%, 103.75% ± 4.27%, 96.64% ± 11.19%, 80.07% ± 4.84%, respectively. Also, there was no significant difference (P > 0.05) in the survival rate of the strain YT155 exposed to pH 6.0, 8.0, and 9.0 compared to the control group (pH 7.0), respectively, suggesting that the viability of strain YT155 was not markedly affected by these pH treatments (pH 6.0–9.0) (Fig. 2A). In addition, the probiotic L. rhamnosus GG could survive and proliferate at gastric acid pH[45], indicating no loss of viability in gastric juice over the 4 h in the pH range of 3.0–7.0[46]. However, survival rates of below 5% were observed when Lactobacillus sp. YT155 and L. rhamnosus GG were exposed to pH 2.0, 3.0, and 4.0, respectively, which may be attributed to the survival rates evaluated through determining the growth by OD600nm value rather than plate counting of bacterial cells surviving in a specific pH condition[47]. The survival rates of Lactobacillus sp. YT155 exposed to pH conditions including 2.0, 3.0, and 5.0 to 9.0 showed no significant difference (P > 0.05) compared with that of L. rhamnosus GG, indicating that strain YT155 possessed a similar pH tolerance to strain LGG. In addition, the adaptability of strain Lactobacillus sp. YT155 to a temperature range of 37°C − 50°C was observed (Fig. 2B). An appreciable growth of Lactobacillus sp. YT155 at 40°C and 50°C was observed, with survival rates of 92.95% ± 0.46% and 84.82% ± 1.35%, respectively. There was a significant difference (P < 0.05) between the treatment of 50°C and the control group (37℃). Moreover, the survival rate of L. rhamnosus GG exposed to temperature above 60℃ was significantly decreased. Normally, the viability of LAB is unaffected by temperatures below 55°C, but they become prone to harm at temperatures above 60°C[48]. In addition, tolerance to the osmotic concentration of NaCl is an important characteristic for the application of probiotics in industries[49]. The tolerance of strain YT155 to NaCl at different concentrations was depicted as shown in Fig. 2C, showing that it could survive in a wide range of salinities from 2–10%. With exposure to 2%, 4%, and 6% NaCl, the survival rates of Lactobacillus sp. Y155 were 126.26% ± 3.30%, 92.91% ± 0.46%, and 41.00% ± 3.69%, respectively. Particularly, the proliferation of strain YT155 was significantly (P < 0.01) enhanced when exposed to 2% NaCl, and its survival rate was superior to that of strain LGG (P < 0.05). Therefore, the results of tolerance of Lactobacillus sp. YT155 to environmental stress indicated it could survive in different environmental conditions including temperature from 37℃ to 50℃, pH from 5.0 to 10.0, and salinity from 2–6%.
The beneficial effects of probiotic strains are also dependent on their ability to survive against natural host defenses and to multiply in the gastrointestinal tract (GI)[50]. Therefore, good tolerance to bile salts, low pH, and digestive enzymes in the GI tract is one of the most important properties for probiotic strains, which allows them to survive in GI. As shown in Fig. 2D, the survival rate of Lactobacillus sp. YT155 was 80.31% ± 9.78% with exposure to 0.1% bile salts, which was similar to that of L. rhamnosus GG with a survival rate of 64.14% ± 5.94% (P > 0.05). Furthermore, a decreasing trend in survival rates of Lactobacillus sp. YT155 was observed with an increase of bile salt concentrations, indicating that high levels of bile salt were detrimental to its survival. Although the concentration of bile salts in the animal intestine varies over time and with the different segments of the intestine, the average concentration is believed to be 0.3% (w/v)[51]. Therefore, the tolerance to 0.3% bile salt was a key criterion to evaluate the capability of Lactobacillus sp. YT155 to survive in the GI tract. Although Lactobacillus sp. YT155 exhibited a survival rate of 6.04% ± 3.53% when exposed to 0.3% bile salt which was lower than that of L. rhamnosus GG (18.24% ± 3.17%), the tolerance of strain YT155 showed no significant difference (P > 0.05) in comparison to that of strain LGG. Therefore, it was speculated that Lactobacillus sp. YT155 possessed the same ability to tolerate 0.3% bile salts as L. rhamnosus GG, showing good potential for survival in the GI tract. In addition, the tolerance to simulated gastric fluid and intestinal fluid in vitro was displayed in Fig. 2E. Lactobacillus sp. YT155 treated with gastric juice (pH 3.0) for 3 h was found to maintain a good survival rate of 83.60% ± 15.09%, with no significant difference (P > 0.05) compared with that of L. rhamnosus GG, suggesting that strain YT155 possessed the good capability to tolerate pepsin and low pH of GI. In addition, the survival rate of Lactobacillus sp. YT155 was 56.98% ± 3.37% after 4 h of incubation in simulated intestinal fluid, indicating that strain YT155 was sensitive to the trypsin to some extent. The first obstacle for a probiotic to enter the GI tract of the host is the acidic environment in the stomach (pH < 2), but the pH of the stomach was elevated to pH 3 due to the buffering effect of ingested food. Therefore, pH 3 is generally regarded as an ideal pH for a successful probiotic to survive[52]. Furthermore, a large number of digestive enzymes were required in the intestine to digest food, so tolerance to different kinds of enzymes is another key parameter for the evaluation of probiotic properties. In conclusion, Lactobacillus sp. YT155 exhibited high tolerance to pepsin, trypsin, and low pH, suggesting that it possessed the potential to survive and colonize in the GI tract.
The ability of probiotics to antagonize pathogens is an important guarantee of survival in the GI tract and maintaining healthy flora [50]. Therefore, the antimicrobial activity of LAB is currently another important selection factor for effective novel probiotics[53]. The inhibition zone diameters of Lactobacillus sp. YT155 against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923 were 21.33 ± 0.58 mm and 14.00 ± 0.00 mm, respectively, showing a good inhibitory effect on foodborne pathogens. As shown in Fig. 2F, there was a significant difference (P < 0.0001) in the inhibition diameters of Lactobacillus sp. YT155 against Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922, suggesting that strain YT155 possessed different inhibition effects on different pathogenic bacteria. In addition, L. rhamnosus GG could produce bactericidal substance that was highly effective against gastric or enteric pathogens of growth[45]. The inhibition zone diameters of Lactobacillus sp. YT155 showed no significant difference (P > 0.05) in comparison to that of L. rhamnosus GG, implying that strain YT155 possessed similar antibacterial activity as strain LGG. Therefore, the antibacterial activity of Lactobacillus sp. YT155 might be attributed to the formation of organic acids, diacetyl compounds, hydrogen peroxide, and bacteriocin-like peptides[54]. In conclusion, Lactobacillus sp. YT155 exhibited good capability to antagonize gastric or enteric pathogens, indicating the potential ability to be used as a candidate probiotic.
The antioxidant ability of probiotics has been demonstrated to be an important characteristic for their preventive role in certain disease conditions like diabetes, heart diseases, and inflammation in intestinal tracts[55]. In addition, probiotics have been demonstrated extensively to help restore the balance of gut microbiota destroyed by oxidative stress[56]. Therefore, antioxidant activity is an important characteristic of an ideal probiotic. The results of scavenging DPPH and hydroxyl radicals by fermentation supernatant and bacteria suspension of Lactobacillus sp. YT155 were displayed in Figs. 2G–2H. The DPPH and hydroxyl radicals scavenging activity by bacteria suspension of Lactobacillus sp. YT155 (1.48% ± 4.82% and 1.38% ± 2.21%) was also similar with that of L. rhamnosus GG (4.91% ± 6.60% and 3.73% ± 1.39%), with no significant difference (P > 0.05). In contrast, the DPPH and hydroxyl radicals scavenging capabilities of the fermentation supernatant of Lactobacillus sp. YT155 were 100.39% ± 1.82% and 34.09% ± 1.57%, respectively, which were close to that of L. rhamnosus GG (97.56% ± 2.43% and 32.41 ± 1.70%) with no significant difference (P > 0.05), which might be attributed to the structure and compositions of the metabolites secreted outside the cells[57]. Although LAB strains were considered as aerotolerant organisms, their oxygen sensitivity is a major factor limiting their viability [58]. Reactive oxygen species (ROS) in cells, such as hydrogen peroxide (H2O2) and highly reactive hydroxyl radicals (HO·), etc., are the main causes of systematic oxidative stress of anaerobes, causing the damage of protein, DNA, and lipid damage, and even cell death[58, 59]. Therefore, Lactobacillus sp. YT155 needs to cope with the oxygen and oxygen-derived free radicals during storage and in the GI tract to maintain high bacterial viability[60]. In addition, young animals are susceptible to oxidative damage because their intestines lack a mature antioxidant system, resulting in an imbalance between oxidative and antioxidant systems, as well as an increase in the free radicals malondialdehyde (MDA) and a decrease in the capacity of antioxidant enzyme[61, 62]. Therefore, Lactobacillus sp. YT155 possessed the potential capability to exhibit substantial antioxidant activity in the host intestine to aid in the removal of ROS in the host intestine and thereby alleviate oxidative damage[58].
Antibiotic resistance was classified into intrinsic and acquired resistance, whose transfer among microbes is one of the major causes of therapeutic failure in infections[63]. The emergence of antibiotic resistance among pathogens and the difficulty in treating infections caused by these bacteria are both possible consequences of LAB acting as a reservoir of transferable antibiotic-resistance genes[64]. Therefore, sensitivity to antibiotics is a prerequisite for probiotic selection. The antibiotic susceptibility of Lactobacillus sp. YT155 was evaluated by using ten common antibiotics, as shown in Table 3. The result showed that Lactobacillus sp. YT155 was sensitive to compounds sulfamethoxazole, ciprofloxacin, and chloroamphenicol, and sensitive-intermediate to six antibiotics including norfloxacin, penicillin, cefamezin, amikacin, gentamicin and ampicillin, and resistant towards erythromycin. Susceptibility to one or the other of the antibiotics indicates susceptibility to all others within the group[65]. Therefore, strain YT155 was susceptible to sulphonamides, quinolones, and chloram phenicols based on the antibiotic susceptibility test. In addition, resistance to antibiotics in lactic acid bacteria is not a cause for worry when the resistance phenotype is caused by mutation or internal resistance mechanisms. Lactobacilli have been demonstrated to be resistant to a variety of antibiotics, including aminoglycosides (e.g., kanamycin, streptomycin, gentamicin), glycopeptides (e.g., vancomycin), and nucleic acid synthesis inhibitors (e.g., ciproflfloxacin)[66], which may be useful for re-establishing the gut microbiota of animals after antimicrobial treatment[67]. Furthermore, transferable resistance genes were not found based on the annotation of genomic sequence of strain YT155 against the Resfinder database. Therefore, it was speculated that Lactobacillus sp. YT155 with resistance to erythromycin could be co-administrated with erythromycin therapy to ensure the replenishment of the healthy gut flora[68]. In conclusion, Lactobacillus sp. YT155 was sensitive to ten commonly used antibiotics with no transferable resistance gene of antibiotics, exhibiting the potential ability to be a safe candidate probiotic.
Table 3
Antibiotic resistance phenotype of Lactobacillus sp. Y155
Antibiotic
|
Zone diameter ± SD (mm)
|
Susceptibility
|
Group
|
Name
|
Concentration (mg)
|
Quinolones
|
NOR
|
10
|
17.33 ± 0.58
|
I
|
Sulphonamides
|
SXT
|
23.75/1.25
|
19.33 ± 0.58
|
S
|
Quinolones
|
CIP
|
5
|
24.00 ± 1.00
|
S
|
Chloram phenicols
|
C
|
30
|
24.67 ± 0.58
|
S
|
Macrolides
|
E
|
15
|
11.57 ± 1.15
|
R
|
Penicillins
|
P
|
10a
|
15.33 ± 0.58
|
I
|
Cephalosporins
|
CZ
|
30
|
17.00 ± 0.00
|
I
|
Aminoglycosides
|
AK
|
30
|
18.00 ± 0.00
|
I
|
Aminoglycosides
|
GM
|
10
|
18.00 ± 1.73
|
I
|
β-Lactams
|
AM
|
10
|
17.33 ± 0.58
|
I
|
The antibiotics used in the present study were norfloxacin (NOR), compound sulfamethoxazole (SXT), ciprofloxacin (CIP), chloroamphenicol (C), erythromycin (E), penicillin (P), cefamezin (CZ), amikacin (AK), gentamicin (GM), and ampicillin (AM), respectively. The strain YT155 with a zone of inhibition (ZOI) less than or equal to 14 mm, more than 20 mm diameter, and between 15 and 19 mm was considered as resistant (R), susceptible (S), and intermediate (I) respectively. In addition, the small letter (a) indicated the concentration of penicillins in U (units).
|
3.3 Genomic features of Lactobacillus sp.YT155
The circular genomic map of YT155 was shown as Fig. 3A. Whole-genome sequencing on the Illumina platform yielded the complete genome sequences for strain YT155 with a size of 1,525,305 bp and the GC content of 34.84%. A total of 1,530 coding sequences (CDSs), 45 tRNAs, and 3 rRNA operons were predicted in the genomic sequence of Lactobacillus sp. YT155. These CDSs were annotated by blasting genes against both COG and KEGG databases. As a result, these 1530 genes were assigned to four main COG categories (metabolism, cellular processes and signaling, information storage and processing, and poorly characterized), which were further allocated to 19 functional groups (Fig. 3B). A total of 1312 CDSs (1312/1530, 85.75%) were classified into the category of function unknown (24.92%, 327/1312), followed by carbohydrate transport and metabolism (11.13%, 146/1312), translation, ribosomal structure and biogenesis (10.52%, 138/1312), and transcription (8.23%, 108/1312). From the KEGG analysis, a total of 912 genes were successfully annotated, and they were assigned to the top five categories including global and overview maps (29.17%, 266/912), carbohydrate metabolism (13.16%, 120/912), membrane transport (11.62%, 106/912), translation (8.88%, 81/912), and nucleotide metabolism (5.48%, 50/912), respectively (Fig. 3C). According to the KEGG and COG classifications, most of the genes in the genomic sequence of strain YT155 are organized in carbohydrate metabolism, implying a relative importance of carbohydrate utilization. Furthermore, the automated functional annotation against the CAZyme database indicated that the genomic sequence of strain YT155 contained 41 putative genes that have been proposed to encode CAZymes, such as carbohydrate esterase (CE), glycosyl transferase (GT), glycoside hydrolase (GH), and auxiliary activity (AA). In particular, GTs (46.34%, 19/41) and GHs (31.71%, 13/41) were the dominant CAZyme categories. GT2 (42.11%, 8/19) and GT4 (31.58%, 6/19) were the two most abundant GTs containing multiple enzymes related to cell wall synthesis. Multiple glycoside hydrolases including beta-xylosidase (EC 3.2.1.37) belonging to GH1 family (46.15%, 6/13) were the dominant GHs in the genomic sequence of strain YT155. Additionally, gene0538 and gene0748 encoded CAZymes belonging to GH43_11 family (Table S2). Up to date, beta-xylosidases, which are the main enzymes responsible for hydrolysis from non-reducing ends of xylo-oligosaccharides and xylobiose to liberate monosaccharide[69], are divided into eleven different groups, including GH family 1, 3, 5, 30, 39, 43, 51, 52, 54, 116, and 120[70]. In particular, GH43 family has been divided into 37 subfamilies, including GH43_11 family comprising xylosidase [EC 3.2.1.37] and arabinofuranosidase [EC 3.2.1.55] for aiding in the degradation of xylans[71]. In addition, GH43_11 family limited to the Ascomycota didn’t contain a signal peptide directing the translated protein outside the cytoplasm, suggesting that this subfamily is involved in intracellular processes, such as the degradation of imported disaccharides or cell wall remodeling[71]. Therefore, it could be speculated that these putative glycoside hydrolases might be involved in the intracellular degradation and metabolism of XOS by strain YT155.
3.4 Genetic analysis of XOS consumption by Lactobacillus sp. YT155
Based on the whole-genome annotation of strain YT155, one 8.4 kb gene cluster (gene cluster no.1) comprising six genes (gene0535, gene 0536, gene0537, gene0538, gene0539, and gene0540) and another 5.5 kb gene cluster (gene cluster no.2) consisting of five genes (gene0748, gene0749, gene0750, gene0751, gene0752) were predicted to link to XOS consumption (Table 4). Gene cluster no.1 consists of xynB (gene0538, designated xylC), xylA (gene0539), xylB (gene0540) encoding putative beta-xylosidase [EC 3.2.1.37], xylose isomerase [EC:5.3.1.5] and xylulokinase [EC:2.7.1.17], respectively, and other three genes encoding a xylose repressor belonging to ROK family protein (gene0536, designated as xylR ) and two MFS transporters including D-xylose transporter (gene0535, designated as xylT) and MFS/sugar transport protein (gene0537, designated as xylP), respectively, as shown in Fig. 4A. The gene xynB (gene0538) spanning 1641 bp encoded a beta-xylosidase (EC 3.2.1.37) containing 546 amino acids, with a C-terminal concanavalin A-like domain. This beta-xylosidase was encoded by xynB exhibiting 72.93% and 71.82% amino acid sequence identity with that of Levilactobacillus koreensis JCM 16448 (Genbank accession no. KRK91779.1) and Levilactobacillus brevis BSO 464 (Genbank accession no. AJA80631.1), respectively. This gene cluster carries a xylose operon (xylAB) involved in the metabolism of xylose through converting cytoplasmic D-xylose to D-xylulose-5-phosphate, an intermediate of the pentose phosphate pathway[72]. In particular, given that these genes are always adjacent to each other, the organization of xylA and xylB seems to be highly conserved in all bacteria [72]. The ROK family protein encoded by xylR (gene0536) was annotated as xylose repressor based on the swiss-prot database, showing 40.99% and 42.75% sequence identity with XylR family transcriptional regulator of Lactiplantibacillus pentosus (Genbank accession no. AUI80112.1) and xylose repressor of Paucilactobacillus hokkaidonensis JCM 18461 (Genbank accession no. BAP86420.1), respectively, which possessed the typical function of ROK-family regulator and the opposite function of XylR of Lactobacillus pentosus MD353[72, 73]. Major facilitator superfamily (MFS) transporters are essential for the movement of a wide range of substrates across biomembranes [74], including the transport of β-galacto-oligosaccharides[75], xylose[76], and XOS[77]. Therefore, we speculated that the two MFS transports encoded by xylT (gene0535) and xylP (gene0537) in gene cluster no.1 might participate in metabolizing XOS.
Table 4
Genetic analysis of XOS consumption by Lactobacillus sp. Y155
Gene cluster
|
Gene ID
|
Gene name
|
Annotation
|
Gene length / bp
|
CAZyme annotation
|
Gene cluster no.1
|
gene0535
|
xylT
|
sugar porter family MFS transporter / D-xylose transporter
|
1347
|
-
|
gene0536
|
xylR
|
a xylose repressor belonging to ROK family protein
|
1161
|
-
|
gene0537
|
xylP
|
MFS/sugar transport protein
|
1452
|
-
|
gene0538
|
xynB, designated as xylC
|
putative beta-xylosidase [EC 3.2.1.37]
|
1641
|
GH43_11
|
gene0539
|
xylA
|
xylose isomerase [EC:5.3.1.5]
|
1338
|
-
|
gene0540
|
xylB
|
xylulokinase [EC:2.7.1.17]
|
1509
|
-
|
Gene cluster no.2
|
gene0748
|
xynB, designated as xylC
|
beta-xylosidase [EC 3.2.1.37]
|
1659
|
GH43_11
|
gene0749
|
xylT
|
MFS transporter
|
1242
|
-
|
gene0750
|
xylF
|
ABC transporter substrate-binding protein
|
993
|
-
|
gene0751
|
xylG
|
branched-chain amino acid ABC transporter permease
|
879
|
-
|
gene0752
|
xylH
|
ATP-binding cassette domain-containing protein
|
753
|
-
|
In addition, gene cluster no.2 (Fig. 4B) consists of xynB (gene0748, designated as xylC) encoding beta-xylosidase [EC 3.2.1.37], gene0749 (designated as xylT) encoding MFS transporter, three genes including gene0750 (designated as xylF), gene0751 (designated as xylG) and gene0752 (designated as xylH) encoding ABC transporter substrate-binding protein (transporter), branched-chain amino acid ABC transporter permease (permease) and ATP-binding cassette domain-containing protein (ATP-binding protein) respectively. The putative beta-xylosidase containing C-terminal concanavalin A-like domain consists of 552 amino acids encoded by xynB with a size of 1659 bp, which showed the 77.94% and 66.18% amino acid sequence identity with that of Paucilactobacillus hokkaidonensis JCM 18461 (Genbank accession no. BAP85153.1) and Limosilactobacillus fermentum MTCC 8711 (Genbank accession no. EQC58821.1) respectively. Also, the beta-xylosidase encoded by the gene cluster no.2 showed low similarity (32.86%) of amino acid sequence with the one encoded by the gene cluster no. 1. The ATP-binding cassette transporter (ABC) sugar system has been reported to be responsible for transporting XOS into the cytoplasm in bifidobacteria by binding it on the cell surface[78]. The ABC transporter system encoded by three genes of gene cluster no. 2 was similar with that of B. animalis subsp. lactis BB-12, consisting of transporter, permease and ATP-binding protein[79]. Therefore, the ABC transporter was speculated to be involved in XOS consumption by strain YT155.
Based on the whole-genome analysis of Lactobacillus sp. YT155, two gene clusters including xylABCPRT and xylCFGHT might be involved in the XOS metabolism. Therefore, a possible pathway of XOS consumption by strain YT155 was proposed to be as shown in Fig. 4C, which was similar with that of Bifidobacterium animalis subsp. lactis BB-12[79]. Firstly, XOS was transported across the cell membrane by ABC oligosaccharide transport system or MFS transporter into the cytoplasm and further hydrolyzed by intracellular beta-xylosidase to produce xylose; then D-xylose was converted to D-xylulose by xylose isomerase, and finally D-xylulose was phosphorylated by xylulose kinase to produce D-xylulose 5-phosphate (X5P), entering the tricarboxylic acid cycle. In particular, there was a xylose repressor belonging to ROK family to regulate the metabolism and utilization of XOS by strain YT155, which was in consistent with the previous study[78]. To date, the XOS metabolism by bifidobacteria has been extensively studied, while the molecular mechanism of XOS metabolism for the species Lactobacillus sp. is largely unknown[80]. The XOS metabolic route of Lactobacillus sp. YT155 in the present study was preliminarily speculated based on the whole genome sequencing, which need to be further verified through transcriptomics and gene knockout experiments, etc.