Achieving High Yield of Lactic Acid for Antimicrobial Characterization in Cephalosporin-Resistant Lactobacillus by the Co-Expression of theosphofructokinase and Glucokinase

Abstract

Lactobacilli are universally recognized as probiotics that are widely used in the adjuvant treatment of inflammatory diseases, such as vaginitis and enteritis. With the overuse of antibiotics in recent years, the lactobacilli in the human body are killed, which could disrupt the microecological balance in the human body and affect health adversely. In this work, cephalosporin-resistant Lactobacillus casei RL20 was obtained successfully from the feces of healthy volunteers, which possessed a stable genetic set. However, the shortage of lactic acid (72.0 g/l at 48 h) by fermentation did not meet the requirement for its use in medicine. To increase the production of lactic acid, the functional genes pfk and glk were introduced into the wild strain. A yield of 144.2 g/l lactic acid was obtained in the transgenic L. casei RL20-2 after fermentation for 48 h in 1 L of basic fermentation medium with an initial glucose concentration of 100 g/l and increasing antibacterial activity. These data suggested that L. casei RL20-2 that exhibited a high yield of lactic acid may be a potential probiotic to inhibit the spread of bacterial infectious diseases and may be used for vaginitis therapy.

Introduction

Lactobacillus is a functional group of microorganisms that are gram positive and found mainly in the intestinal tract and the vagina [22]. Lactobacillus, as a probiotic, affects human beings significantly. It has been widely used in animal husbandry and food processing. Through research, Lactobacillus was applied to treat diseases by enhancing the ability of a patient’s immunity and antagonistic effect to pathogens and improving the body’s immunity and its maturity, as well as nutrition and growth stimulation [8,11]. Lactobacillus inhibits pathogens mainly because it secretes lactic acid. The acidic environment caused by the lactic acid of Lactobacillus helps to reduce the negative charge on the surface of cells and remove the sugar group covered on the surface of the receptor, which could inhibit the growth of pathogens and maintain the balance of the microenvironment in the human body [17,25]. In addition, Lactobacillus was also reported to treat inflammatory bowel disease and gynecological diseases [24]. Lactobacillus, as a probiotic, possesses a wide range of functions. However, the application of bacteria to the vagina has many challenges. One of the major threats is that Lactobacillus is killed simultaneously when antibiotics are used during the treatment of bacterial diseases.

Clinically, antibiotics such as metronidazole, lincomycin, and cephalosporins are the most widely used drugs to treat vaginitis [18], and these drugs were mostly effective. Nevertheless, the therapies for vaginal infections in women have changed only slightly; thus, finding a cure is more difficult because of the reduction of lactobacilli and the disruption of the vaginal environment [21,23]. Hypothetically, we can detect stable and genetically resistant Lactobacillus from the human intestinal tract, which can resist cephalosporin, an ingredient of most popular drugs to treat vaginitis. When patients take medications with cephalosporin, this type of Lactobacillus will survive and even secrete lactic acid to maintain the microenvironment condition in the body, thus reducing the antagonism to the vagina caused by antibiotic therapy.

The antibacterial ability of Lactobacillus depends mainly on the secretion of lactic acid; thus, it must secrete high amounts of lactic acid [32], thereby making this probiotic valuable. At present, wild strains of Lactobacillus rarely produce a high amount of lactic acid, whether it was used for food or drugs. Numerous studies were conducted to acquire a relatively high yield of lactic acid by improving the fermentation conditions. However, few studies aim to increase lactic acid yield while regulating the glycolytic flux in Lactobacillus. Until now, the D-lactate productivity increased individually and through the simultaneous overexpression of five glycolytic genes coding for glucokinase (GLK), glyceraldehyde phosphate dehydrogenase (GAPDH), phosphofructokinase (PFK), triosephosphate isomerase (TPI), and bisphosphate aldolase (FBA) [27,28]. For gene modification, transforming the least gene is a good strategy to achieve a satisfactory lactic acid production, which may reduce the uncertain factors that may adversely affect the experiment and improve its reliability. Hence, we can increase the yield of lactic acid through the co-expression of PFK and GLK, which are closely related to its manufacture. In this study, a cephalosporin-resistant Lactobacillus casei strain was screened from human feces; a good yield of lactic acid was obtained, and the antibacterial ability increased after the genetic modification. Thus, this bacterium could be used as a potential drug to treat infectious diseases, such as vaginitis and enteritis.

 

Materials and Methods

Design of the Oligonucleotide Microarray

The oligonucleotide-based DNA microarray from MWG Biotech (NC, USA) had 39 bacterial species and 22 antibiotic-resistant genes according to the microarray by Wang et al. [30]. The bacterial DNA and antibiotic-resistant genes assayed in this study are listed in Tables 1 through 3. The bacteria were derived from the 16S rRNA gene database (the Entrez nucleotide database), whereas the antibiotic-resistant genes were selected according to the Antibiotic Resistance Genes Database. Three different oligonucleotides for each gene were situated apart from each other within the protein coding sequence and were represented in the microarray in an 18 × 18 format. Each oligonucleotide consisted of 26 to 37 basepairs with similar physicochemical parameters. The specificity of the GeneChip can detect as low as a single nucleotide difference between oligonucleotides. Thus, species-specific probes could be designed for each species.

Table 1.Phylogenetic distribution of bacterial phylospecies from human intestine that can be interrogated by microbiota array.

Table 2.List of Lactobacillus and Bifidobacterium bacteria presented in the microbiota array.

Table 3.F represents a primer of 5’terminal for a gene, and R represents a primer of 3’terminal for a gene.

DNA Hybridization and Detection with the Oligonucleotide Microarray from Fecal Samples

Two grams of fresh feces obtained from four healthy volunteers of two different age groups (two children and two adults) was resuspended in 8 ml of PBS, and the chromosomal DNA was extracted from 400 μl of the fecal slurry by using the QIAamp DNA Stool Mini Kit (Qiagen, CA, USA). The bacteria universal primers for 16S rDNA were F:5’-AGRGTTYGATYMTGGCTCAG- 3’ and R:5’-GGYTACCTTGTTACGACTT-3’ (Y is C or T; R is A or G; F is a primer of 5’terminal for a gene; R is a primer of 3’terminal for a gene) [30,31]. The primers for antibiotic-resistant genes are indicated in Table 3. PCR amplification with CY5-dCTP was performed by using a GeneAmp 9700 PCR system (PerkinElmer, CT, USA), and the PCR program was set t o 95℃ for 3 m in followed by 30 cycles of 95℃ for 10 sec, 53℃ for 10 sec, and 72℃ for 70 sec, with a final 4 min extension at 72℃. The Cy5-labeled PCR product was heated in a boiling water bath for 3 min and immediately cooled in ice water before being used.

After PCR amplification, the chromosomal DNA was dissolved in 15 ml of MWG Biotech hybridization buffer. The tube was heated for 3 min in a boiling water bath and immediately placed in ice water for 2 min. The solution was collected by centrifuging the sample at 3,000 ×g and applied onto the oligo area on the microarray slides. A small glass cover slip that was autoclaved and dried was used to cover the hybridization solution on the array area. The slide was placed in a hybridization chamber (Corning Inc., NY, USA) and immersed in a water bath for hybridization overnight at 42℃. The successfully hybridized microarray was washed twice for 5 min with 0.5× SSC and 0.1% SDS. Afterwards, the microarray was dried by centrifugation for 1 min at 3,000 ×g and scanned with the ScanArray Express Microarray Scanner (Packard BioScience-Perkin Elmer, USA).

Screening of Lactobacillus and Bifidobacterium Species from Fecal Samples with the Selective Agar Slab

Fresh fecal samples were harvested from four healthy volunteers and diluted to serial dilutions. The dilutions were coated with the MRS and TPY agar medium that contained 30 μg of antibiotic (cefoxitin, cefotaxime, cefepime) and incubated anaerobically at 35.8℃ for 24 h. The single colonies were taken out and streaked to purify the MRS broth media (for lactobacilli) or improve the MRS broth media, which contained 0.2% sodium sulfide and bile salt (for bifidobacteria) at 35.8℃ for 24 h. The resulting strains were characterized by Gram staining and analyzed by using the Microbial Identification System (MIDI Inc., DE, USA). The positive strains from culture plating were selected and sequenced at Shanghai Invitrogen Company, China.

Construction of Recombinant Lactobacillus casei RL20

The total DNA was extracted from L. casei RL20 with QIAamp Fast DNA Stool Mini Kit (Qiagen). Genes pfk and glk were amplified with ExTaq DNA polymerase (Takara), dNTP (N is A or T or C or G), and primers (shown in Table 4). PCR amplification was performed with the same process as above. The resulting pfk and glk were linked into pMG36e to yield recombinant plasmids pMG36e-glk and pMG36e-pfk-glk (the detailed recombinant processes are indicated in Fig. 1) with the modified primers (shown in Table 5). The plasmids were incubated in the mixture with the BP Clonase II Enzyme Mix (Invitrogen) at 25℃ for 4 h and transformed to competent L. casei RL20 cells by using the Gene Pulser Apparatus (BioRad, CA, USA) according to the manufacturer’s instructions. Thus, L. casei RL20-1 and RL20-2 possessed pMG36e-glk and pMG36e-pfk-glk, respectively. The positive bacteria were selected on MRS agar medium that contained erythromycin (200 μg/ml).

Table 4.Bold font represents the restriction site of the enzyme.

Table 5.Bold font represents the restriction site of the enzyme; underlined letters indicate the introduced restriction site of the enzyme.

Fig. 1.Construction of recombinant plasmids pMG36e-glk and pMG36e-pfk-glk.

Enzyme Assays with Lactobacillus casei RL20, RL20-1, and RL20-2

The enzyme assays were performed using a UV spectrophotometer (TU-1810S; Persee, China) at 340 nm by monitoring the decrease or increase of NAD(P) or NADH. One milliliter of the reaction solution of L. casei RL20, RL20-1, and RL20-2 was harvested by centrifuging the sample at 10,000 ×g, 4℃ for 5 min. The cells were resuspended in 1 ml of sonication buffer with 0.5 g glass beads, and the suspensions were sonicated for 45 min with an ultrasonic homogenizer (UCD-200; Cosmo Bio, Japan) in an ice water bath. The supernatant was analyzed to determine GLK, GAPDH, PFK, TPI, FBA, and LDH (lactate dehydrogenase) based on the method of Tsuge et al. [27]. GLK activity was measured in 100 mM Tris-HCl buffer (pH 7.5) that contained 1 U/ml glucose-6-phosphate dehydrogenase. GAPDH activity was measured in 25 mM sodium phosphate and 25 mM triethanolamine buffer (pH 7.5) that contained 5 mM glyceraldehyde-3-phosphate. PFK activity was measured in 100 mM Tris-HCl buffer (pH 7.5) that contained 10 mM fructose-6-phosphate, 1 U/ml FBA, 5 U/ml TPI, and 5 U/ml glycerol-3- phosphate dehydrogenase. TPI activity was measured in 100 mM Tris-HCl buffer (pH 7.5) that contained 15 mM glyceraldehyde-3-phosphate and 5 mM G3PDH. FBA activity was measured in 100 mM Tris-HCl buffer (pH 7.5) that contained 20 mM fructose-1,6-bisphosphate, 1 U/ml TPI, and 5 U/ml G3PDH. LDH activity was measured in 25 mM MOPS buffer (pH 7.0) that contained 2 mM sodium pyruvate. Then, the supernatant was detected in a UV spectrophotometer with the control group, and the absorbance values were measured every 15 sec. Finally, the enzyme activities were calculated according to the enzyme formula. Units of these activities were calculated using an extinction coefficient for NAD(P)H/NAD(P) of 6,220 M-1 cm-1 at 340 nm.

Determination of Lactic Acid and Growth Curve

Fermentation experiments were carried out in 2 L flask that contained 1 L of the basic fermentation medium (7.5 g/l yeast extract, 15 g/l peptone, 10 g/l beef extract, 0.2 g/l MgSO4·7H2O, 0.05 g/l MnSO4·4H2O, pH 7) with an initial glucose concentration of 100 g/l, and then the glucose concentration in the later fermentation process was maintained above 50 g/l. Flasks were inoculated with 5% inoculum culture and incubated in a shaker incubator at 42℃ with shaking at 150 ×g. The culture supernatant was harvested at various time intervals and analyzed for lactic acid, and the growth curve was established by utilizing a high-performance liquid chromatography system (8020; Tosoh Corp., Japan) equipped with a 210 nm UV detector. The column was COSMOSIL5C18-PAQ (4.6 mm × 250 mm, 5 μm), the mobile phase was 0.2 mol/l KH2PO4 solution plus 1% acetonitrile, and the pH value was adjusted from 2.6 to 2.8.

Detection of Antibacterial Activity of Lactobacillus Strains

The harvested Lactobacillus strains grown on MRS broth were suspended in 0.9% NaCl until the optical density (OD) of the suspension at 600 nm was 0.4. Plates with a diameter of 4 cm and that contained 15 ml of MRS agar were inoculated with 200 μl of lactobacilli and incubated at 37℃ and 5% CO2 for 24 h. Agar slabs with a diameter of 9 mm were cut and placed on agar inoculated with 0.5 ml of the target indicator strain suspended in 0.9% NaCl (OD600 = 0.1). To obtain the initial diffusion of the substance from the agar slabs, the plates were first refrigerated for 4 h at 4℃ and kept for 20 h at 37℃ in aerobic conditions. After incubation, the plates were checked for inhibition zones.

Statistical Analysis

The data were evaluated by using GraphPad Prism 5.0. Statistical analysis for significant differences was performed by using oneway ANOVA when appropriate. The data are presented as the mean. Error bars represent the standard deviation of the means for at least three independent experiments. A p-value < 0.05 was considered statistically significant.

 

Results

Validation of the Designed Oligonucleotide Microarray

The validation of the microarray was conducted with 10 bacterial strains (purchased from American Type Culture Collection (ATCC)), including Bacteroides thetaiotomicron ATCC 29148, Clostridium clostridioforme ATCC 29048, Fusobacterium russii ATCC 25533, Ruminococcus gnavus ATCC 29149, Bifidobacterium infantis ATCC 15697, Bifidobacterium bifidum ATCC 29521, Lactobacillus fermentum ATCC 9338, Lactobacillus acidophilus ATCC 4796, Escherichia coli ATCC 25922, and Finegoldia magnus ATCC 14955, and seven antibiotic resistance genes (synthesized by Generay Company), aac(6’)-Ii, mecA, blaZ, catpC194, mph(C), tet(k), and van(E), as listed in Table 6. A total of 250 ng of CY5-labeled chromosomal DNA was hybridized with the array at 42℃ overnight. The distribution layout of oligonucleotides on the microarray are shown in Fig. 2, and the results are presented in Fig. 3. Nine strains were reliably detected (with at least 96% confidence) by the array except for E. coli ATCC 25922. Its weak signal was due to the significantly lower amount of viable organisms under anaerobic conditions, which may lead to a quantity of chromosome DNA that is lower than the detection threshold (4 ng) of the array. However, the array could correctly discriminate bacteria among closely related species within the same genus, as indicated by the results. A negative control array was run with the standard hybridization mixture without any bacterial DNA except for herring sperm DNA (carrier) and B2 oligomers (hybridization standard). As expected, this scanned microarray was largely blank with no probe sets showing signal values marginally above the level of background noise. Simultaneously, the seven antibiotic resistance genes were all detected correctly (with at least 97% confidence) with strong signal values.

Table 6.Bacteria and antibiotic resistance genes used to validate the microbiota array.

Fig. 2.Distribution layout of the oligonucleotides on the microarray. Details of the detectable genes are given in Tables 1 and 3. The control (Ctrl) consisted of CY5-labeled oligonucleotides.

Fig. 3.Microarray results of the DNA mixture of ten reference bacterial species and seven antibiotic resistance genes. The intensities of signal from high to low are white (saturated), then red, yellow, green, and blue. If two or three probes for one species gave positive signals, the sample was considered positive (+) for this species. If only one or no probe for one species showed a positive signal, the sample was considered negative (-) for this species.

Detection of Bacterial Strains from Fecal Samples with the Designed Oligonucleotide Microarray

Fresh fecal samples were detected with the microarray, and the results of this assay are shown in Fig. 4. Twenty-five bacterial strains, including seven species of Lactobacillius, five species of Bifidobacterium, three species of Clostridium, two species each of Bacteroides and Ruminococcus, and single species each of Fusobacterium, Faecalibacterium, Collinsella, Finegoldia, Eubacterium, and Enterococcus, and 11 antibiotic resistance genes, including bleO, str, blaZ, acrA, bl2_kpc, catD, van(B), van(E), tet(k), tetA(P), and mph(C), were presented in the microarray. Some of the results were consistent because Lactobacillus [5] and Bifidobacterium [16] are the June 2016⎪Vol. 26⎪No. 6 most important bacteria in the human intestinal tract. blaZ, which resisted most clinical cephalosporins, occurred in the microarray. Notably, we exactly verified only blaZ in this assay.

Fig. 4.Microarray test results for the DNA mixture from fresh fecal samples. The species numbers are 1, 3, 7, 11, 14, 15, 17, 18, 20, 21, 25, 27, 30, 34, 35, 36, 42, 43, 48, 53, 63, 68, 72, 75 and 3r, 4r, 6r, 8r, 10r, 15r, 17r, 18r, 19r, 20r, 22r, according to Fig. 2.

Screening of Antibiotic-Resistant Bacteria from Fecal Samples with the Selective Agar Slab

The 11 antibiotic resistance genes presented in the microarray (as shown in Fig. 4) can resist aminoglycosides, β-lactams, phenicols, tetracyclines, glycopeptides, and macrolides in the bacteria. Generally, we focused on cephalosporins in this study, such as cefotaxime and cefepime, which were widely used in clinical tests. Thus, cefoxitin, cefotaxime, and cefepime were selected and applied to MRS agar medium to select the Lactobacillus and Bifidobacterium that contained the cephalosporin-resistant gene. In our study, three bacterial strains (RL1, RL11, and RL20) in MRS and one bacterial strain (RB13) in TPY were obtained. The 16S rRNAs of the four isolated bacteria were identified by agarose gel electrophoresis. The results are indicated in Fig. 5, where a prominent band of approximately 1,500 bp was observed in the strains of RL1, RL11, RL20, and RB13, which matched the calculated molecular weight of 1 6S r RNA of Lactobacillus and Bifidobacterium. This finding suggests that these strains belong to the genus Lactobacillus and Bifidobacterium. They were sequenced and identified as L. brevis RL1, L. acidophilus RL11, L. casei RL20, and B. longum RB13 at Shanghai Invitrogen Company. Thus, the four bacteria could be utilized in further research as candidate strains.

Fig. 5.Amplification of the 16S rRNA of the four isolated bacteria. M: DNA marker DL5000.

Measurement of Lactic Acid from the Four Isolated Bacteria and Glycolytic Enzyme Activities of Lactobacillus casei RL20

Fermentation assays of the four isolated bacteria were carried out at 42℃ with shaking at 150 ×g in the flask. Results (shown in Fig. 6) indicated that the lactic acid increased gradually over time in 48 h. Strain RL1 exhibited the lowest yield of lactic acid at 39.7 g/l. Strain RL20 produced the highest amount of lactic acid at 72.9 g/l. However, the lactic acid yield was evidently lower than the 113 g/l produced by Yu et al. [33]. Hence, we selected strains RL11, RL20, and RB13, which performed better in terms of lactic acid production, for further study.

Fig. 6.Lactic acid yield from L. brevis RL1, L. acidophilus RL11, L. casei RL20, and B. longum RB13 under oxygen deprivation. Data points for lactic acid concentration represent the averages calculated from triplicate measurements. Standard deviations are indicated by bars.

Glycolysis is the fundamental pathway to metabolize glucose to pyruvate, which is a key intermediate metabolite that leads to the production of lactic acid in Lactobacillus and Bifidobacterium under anaerobic condition. In this pathway, GLK, GAPDH, PFK, TPI, FBA, and LDH are relatively close to lactic acid production. We detected these enzymes, and the detection results in strains RL11, RL20, and RB13 are shown in Fig. 7. Generally, the six enzyme activities of all strains increased before 24 h and decreased after 24 h. Thus, the highest activity of the six enzymes all occurred at 24 h. Strain RL20 exhibited the highest activity at 24 h among the three strains, whereas strain RB13 had the least. The GLK activity of strain RL20 was 33.3% higher with 2.4 ± 0.2 U/mg protein at 24 h, compared with 1.8 ± 0.2 U/mg protein of strain RL11. The GAPDH, PFK, TPI, and FBA activities in strain RL20 increased by 59.8%, 45.5%, 21.5%, and 15.9%, respectively, compared with that of strain RB13. The enzyme activity of LDH was still nearly 30% higher in strain RL20 than both strains RL11 and RB13. As shown in Fig. 6, strain RL20 obtained the best lactic acid yield among the three strains. However the amount of lactic acid produced may not meet the application requirements. In these enzymes, the GLK and PFK activities in strain RL20 were 2.4 ± 0.2 and 0.64 ± 0.08 U/mg protein, respectively. These activities were significantly lower than those of the four other enzymes. Alteration was necessary to enhance the lactic acid production by improving the GLK and PFK activities.

Fig. 7.Enzyme activities of GLK (A), PFK (B), GAPDH (C), TPI (D), FBA (E), and LDH (F) in L. acidophilus RL11, L. casei RL20, and B. longum RB13 in the bioprocess reaction. Data points represent the averages calculated from triplicate measurements. Standard deviations are indicated by bars.

Identification of pMG36e-glk and pMG36e-pfk-glk in Lactobacillus casei RL20

Genes pfk and glk were inserted into the pMG36e vector to yield plasmids pMG36e-glk and pMG36e-pfk-glk, which were then transferred into strain RL20 and formed strain RL20-1 that contained pMG36e-glk and strain RL20-2 that contained pMG36e-pfk-glk. The two strains were cultured at 35.8℃ for 24 h in the MRS media. The expression of PFK and GLK was detected by sodium dodecyl sulfate polyacrylamide gel electropheresis. Fig. 8 shows that two slight bands of approximately 34 kDa in the supernatant of strain RL20 were present, which were consistent with the PFK and GLK in L. casei. A tiny band nearly integrated with the thick band was observed in strain RL20-1 where glk was overexpressed, thereby indicating a sharp increase of the GLK enzyme compared with strain RL20. A prominent band of approximately 34 kDa was present in the supernatant because of the co-expression of pfk and glk in strain RL20-2. Thus, we can conclude that recombinant plasmids pMG36e-glk and pMG36e-pfk-glk effectively overexpressed the target gene in the bacteria strain RL20.

Fig. 8.SDS-PAGE analysis of the PFK and GLK. M: Prestained marker. The arrow represents the site of the PFK and GLK in the band.

Biochemical Characteristics of Lactobacillus casei RL20, RL20-1, and RL20-2

We checked the GLK, PFK, and LDH in L. casei RL20, RL20-1, and RL20-2 to investigate the effect of the overexpression of GLK and PFK. Many marked differences were observed in the GLK, PFK, and LDH activity of the three strains after being cultured for 6 h. The GLK activity (indicated in Fig. 9A) exhibited a sharp increase of 6 and 5.7 times higher in strain RL20-1 and strain RL20-2 than that in strain RL20, with 18.3 ± 1.5 and 17.4 ± 1.5 U/mg protein (p < 0.001) respectively. However, the activity reduced dramatically to 5.2 ± 0.8 and 4.1± 0.7 U/mg protein in strain RL20-1 and strain RL20-2 at 48 h, respectively. However, the GLK activity of strain RL20-1 and strain RL20-2 was still twice that of strain RL20. These results clearly proved that the significant increase of GLK activity was facilitated by its overexpression. However, the overexpression of PFK in L. casei RL20-2 did not affect the GLK activity.

Fig. 9.Enzyme activities of GLK (A), PFK (B), and LDH (C) in L. casei RL20, L. casei RL20-1, L. casei RL20-2 in the bioprocess reaction. *Represents p < 0.05 and **represents p < 0.001 in the enzyme activities test against the control group.

The PFK activity (shown in Fig. 9B) increased 17 times at 11.4 ± 1.0 U/mg protein (p < 0.001) in strain RL20-2 compared with that of strain RL20 at 24 h. However, PFK activity exhibited a 2-fold increase in strain RL20-1, which was quite different from the individual overexpression of the GLK. These results suggested that excessive metabolism by the overexpression of the GLK may cause a slight increase in PFK activity, whereas dramatically increased PFK activity occurred with the overexpression of the PFK. However, PFK still maintained a high activity in strain RL20-2 even at 48 h. Therefore, the PFK activity in strain RL20-2 could be enhanced by transferring the pfk and glk.

LDH is a key enzyme that catalyzes pyruvate to lactic acid and can be activated by fructose-1,6-bisphosphate, which is produced by fructose-6-phosphate catalyzed by the PFK enzyme [28]. Our data on LDH activity (shown in Fig. 9C) indicated a half increase in strain RL20-2 at 24 h with 1,601.3 ± 30 U/mg protein (p < 0.001) compared with that of strain RL20 and strain RL20-1. No remarkable changes were observed in strain RL20-1, whereas the same changes occurred in strain RL20-2 at 48 h. Interestingly, the increase of the LDH activity was significantly lower than that of the PFK activity over time. This result may be due to the inhibition of the further activity of LDH by the accumulation of lactic acid. These results clearly demonstrated that the overexpression of PFK was crucial to increase the LDH activity, which was 40% higher than that of the individual overexpression of GLK during fermentation.

We checked the lactic acid concentration and growth curves from the fermentation liquid of L. casei RL20, RL20-1, and RL20-2. Fig. 10 indicates that the lactic acid concentration in the initial stage of bacterial growth was significantly less. When the bacteria reached the logarithmic growth phase, the lactic acid increased significantly in L. casei RL20-2 with 114.9 ± 6.8 g/l (p < 0.001) compared with that of RL20 and RL20-1.When the bacteria reached the stable stage, the OD value of L. casei RL20-2 reduced faster than that of L. casei RL20 because of the accumulation of lactic acid and the consumption of the culture medium. However, a relatively high OD value was maintained, which was equal to that in the logarithmic growth phase and achieved the highest lactic acid yield with 144.1 ± 8.0 g/l (p < 0.001). Results indicated that lactic acid concentration increased, whereas the lactic acid production rate in strain RL20-2 decreased dramatically after 24 h with an average rate of 5.55 g/1-1 h-1 to 1.22 g/1-1 h-1. These results were similar to the previous results of enzyme activity. In summary, strain RL20-2 was structured successfully with the overexpression of GLK and PFK, which can yield twice the amount of lactic acid compared with that of the wild strain.

Fig. 10.Lactic acid concentration (solid icon) and growth curve (hollow icon) with L. casei RL20, L. casei RL20-1, and L. casei RL20-2 under oxygen deprivation. Data points for the lactic acid concentration and growth curves represent the averages calculated from triplicate measurements. Standard deviations are indicated by bars.

Antibacterial Activity of Lactobacillus Strains

The diameter of the growth inhibition zones of the indicator bacteria induced by L. casei RL20, RL20-1, and RL20-2 ranged from 7 to 22 mm, and the diameter of the slab was 9 mm. All three strains inhibited the growth of E. coli, Salmonella sp., S. aureus, Shigella sp., and Pseudomonas aeruginosa (Table 7). The antimicrobial activity of the lactobacilli was correlated with their property of yielding lactic acid. A particularly strong antagonism against all the test bacteria was observed with L. casei RL20-2, which exhibited the highest mean diameters of inhibitory zones with 17.2 ± 1.4 mm (p < 0.05). However, the mean zones with L. casei RL20-1 and L. casei RL20 were 89% and 42% lower than that with the L. casei RL20-2, at 12.1 ± 1.5 and 9.1± 0.9 mm, respectively. This result might be due to the deficiency of lactic acid in strains RL20-1 and RL20, which moderated the acid environment and reduced the antibacterial activity of Lactobacillus. The result indicated that strain RL20-2 effectively inhibited the pathogenic bacteria with strong antagonistic properties.

Table 7.The results are presented as mean diameter of the growth inhibition zone (mm) for three independent experiments; the diameter of the agar slab was 9 mm.

 

Discussion

Antibiotics have been widely used to fight bacterial diseases for more than a hundred years. Although many lives have been saved by antibiotics, many challenges still exist. Antibiotics exhibit a strong killing ability against microorganisms, particularly some extended-spectrum antibiotics, because of the lack of high specificity; thus, both pathogenic and beneficial microorganisms for the human body are killed, thereby changing the microbial flora in the body significantly, which may adversely affect people [3] because probiotics play a functional role in the growth and health of humans [1,7]. Thus, the fact that antibiotic treatment for infectious diseases impairs people’s health is dumbfounding. Otherwise, a major health problem still exists in the use of antibiotics. Alarmingly, the treatment of infections using antibiotics in the long run may result in the difficulty of eliminating pathogenic bacteria. This result is attributed to the emergence of resistant strains because of antibiotic overuse and the powerlessness of infection control measures in hospitals [13], which cause serious threats to global public health. In recent years, several alarming studies indicated that third-generation cephalosporin-resistant strains that produced an extended-spectrum β-lactamase were widespread in many countries, including France [26], China [10], the USA [34], and Japan [2]. A retrospective study demonstrated that the pathogen Escherichia coli that harbored a third-generation cephalosporin-resistant gene increased from 1.2% in 2001 to 19.7% in 2008 in antibiotic consumption; Klebsiella pneumoniae also showed a similar dramatic increasing trend [16]. The same increase was observed by Van der Steen et al. from 2008 to 2012 [29]. Hence, antibiotic-resistant strains could affect the public in the coming decades if no effective control measures are enacted. Moreover, research demonstrated that antibioticresistant strains may be transmitted from food animals to humans [15], and the transferability of extended-spectrum β-lactamase-encoding genes from one species to another [10] facilitated the more rampant diffusion of these pathogens. Two strategies that may limit the emergence of antibiotic resistance strains were highly compliant with infection control measures and strict restrictive use of antibiotics [26]. However, the development of antibioticresistant strains from 2008 to 2012 [29] overcame these strategies decisively, as indicated by later investigation. Thus, constructive work is essential to fight antibioticresistant bacteria.

The genus Lactobacillus is a universally acceptable probiotic that is widely used and occurs naturally in the human intestinal tract and vagina, and confers a fundamental interest on the host [12]; its favorable antagonistic activity against pathogenic microorganisms suggests a valuable biotherapeutic for infectious diseases [5]. Lactobacillus was reported to be susceptible to certain antibiotics, such as ampicillin, gentamicin, erythromycin, tetracycline, penicillin, and vancomycin [5,6]. Exposure to these antibiotics would eradicate Lactobacillus in the human body. In this study, we detected L. casei with a microarray from human feces that carried a third-generation cephalosporin-resistant gene. This screened strain can manufacture cephalosporinase-disabled cephalosporin that protects itself from antibiotics. Administering cephalosporin-resistant strains to patients who consumed cephalosporins would balance the benefit of microbial flora in vivo.

Lactic acid is one of the most important organic compounds of the genus Lactobacillus because of its widespread applications in food, cosmetics, and pharmaceutical and chemical industries [4]. Literature documented that lactic acid and Lactobacillus played a crucial role in vaginal health [9,20]. To be a valuable probiotic or biological medicine, lactobacilli must exhibit high lactic acid production. However, in reality, the production of lactic acid in screened wild Lactobacillus species is often unsatisfactory. Hence, lactic acid production must be improved. Conventionally, studies focused on improving lactic acid production through bacterial fermentation [11]. This strategy could be highly unrealistic for it to be developed as a drug. To increase lactic acid yield, we focused on regulating glycolytic genes. We overexpressed glycolytic genes encoding GLK and PFK in screened Lactobacillus species that closely participate in the manufacture of lactic acid. The result showed that lactic acid production significantly increased (p < 0.001) in RL20-2 after fermenting the sample for 48 h (Fig. 10), as demonstrated by Tsuge et al. [27], which indicated the good capacity of lactic acid production. Subsequently, the experiment to inhibit bacterial growth showed that this engineered Lactobacillus exhibited an increasing antagonism capability (p < 0.05) to the pathogenic bacteria E. coli, Salmonella sp., S. aureus, Shigella sp., and P. aeruginosa (Table 7), thereby suggesting excellent antibacterial activity.

Overall, we successfully screened cephalosporin-resistant L. casei RL20 by microarray. This strain was introduced with pfk and glk to exhibit a strong lactic acid production ability, which exerted a potential value to regulate the microenvironment of the human body, such as the vagina, and inhibit the growth of pathogens. This strain could be developed as a good treatment for women who suffer from bacterial vaginitis and other gynecological inflammations.