36 CHAPTER 4 RESULTS 4.1 Antimicrobial Properties of L. rhamnosus ATCC 7469 CFS The antimicrobial properties of L. rhamnosus ATCC 7469 CFS against P. gingivalis ATCC 33277 were assessed based on two procedures which were disc diffusion assay and broth microdilution assay for susceptibility determination. 4.1.1 Disc Diffusion Assay of L. rhamnosus ATCC 7469 CFS The CFS of L. rhamnosus ATCC 7469 showed high inhibition zone diameter (10.64 ± 0.44 mm) as shown in Table 4.1. The mean inhibition diameter of the positive control, 0.2% chlorhexidine was larger at 16.88 ± 2.16 mm. Meanwhile, as expected the negative control sterile MRS broth showed no inhibition at all. The CFS inhibition diameter were statistically significant in comparison to negative control at p<0.001 with the ANOVA test. Table 4.1: The inhibition diameters of the tested samples against P. gingivalis in disc diffusion assay (significant results are marked by asterisk (*) with p-value<0.001 when being compared to negative control). Test Samples Inhibition diameter, Mean±SD (mm) Significance L. rhamnosusATCC 7469 CFS 10.64 ± 0.44* p-value <0.001 0.2% chlorhexidine 16.88 ± 2.16 Sterile MRS broth - 37 4.1.2 Minimum Inhibitory and Bactericidal Concentration of L. rhamnosusATCC 7469 CFS The MIC and MBC were determined to identify the lowest concentration of L. rhamnosus ATCC 7469 CFS to inhibit P. gingivalis ATCC 33277. The MIC and MBC values of L. rhamnosus ATCC 7469 CFS were identified as 25% (v/v, CFS/sterile broth) (Table 4.2) compared to positive control, 0.2% chlorhexidine at 3.125%. The negative control, sterile MRS recorded no inhibitory and bactericidal effect on P. gingivalis ATCC 33277. The result indicated that the CFS was able to inhibit and simultaneously kill the pathogen at 25% v/v concentration. This was shown in Figure 4.1 where the clear well indicated that there was no growth of P. gingivalis which was marked by the MTT solution. The MTT solution stained the culture with specific metabolic activity related to growth. Meanwhile, the wells that showed colour changes indicated that there was growth of P. gingivalis. The determination of MBC was also demonstrated in Figure 4.1. The visualization of MBC values determination was as shown in Figure 4.2 where MBC was determined by the streak of culture that showed no culture at all or less than 10% colony on the plate. The results indicated that all P. gingivalis colony exposed to the particular concentration of CFS were killed. In this study, 25% v/v concentration of CFS was found to kill more than 90% of P. gingivalis. 38 Table 4.2: The minimum inhibitory and bactericidal concentration of L. rhamnosus ATCC 7469 CFS as determined by broth microdilution assay. Test Samples Minimum Inhibitory Concentration, MIC (%, v/v) Minimum Bactericidal Concentration, MBC (%, v/v) L. rhamnosusATCC 7469 25 25 0.2% Chlorhexidine <3.125 <3.125 Sterile MRS broth >100 >100 Figure 4.1: The representative plates for MIC after being stained with MTT solution. Figure 4.2: The representative streak plate for MBC determination. Sterile MRS 25% L. rhamnosus CFS 0.2% chlorhexidine 39 4.2 Anti-biofilm Properties of L. rhamnosus ATCC 7469 CFS The anti-biofilm properties were determined by measuring the optical density of biofilm that was stained by crystal violet solution in 96-well plate setups. The staining density was measured at 600nm by spectrophotometer to determine the intensity of stained biofilm in each well. The reduction of biofilm percentage varied according to the concentration of L. rhamnosus ATCC 7469 CFS. As shown in Figure 4.7, the highest concentration of L. rhamnosus ATCC 7469 CFS at 100% (v/v, CFS/sterile MRS) recorded the highest percentage of biofilm reduction at 84.94% while the lowest concentration of CFS recorded the percentage of biofilm reduction at 14.00%. Meanwhile, in positive control, the highest concentration of 0.2% chlorhexidine recorded 96.03% percentage of biofilm reduction. On the other hand, in the negative control, sterile MRS broth recorded no biofilm reduction at all. All results for test samples were statistically significant when compared to the untreated biofilm (p-value > 0.001). 40 Figure 4.3: P. gingivalis biofilm inhibition by L. rhamnosus ATCC 7469 CFS. 0.00 20.00 40.00 60.00 80.00 100.00 120.00 100 50 25 12.5 6.25 3.125 P e rc e n ta ge o f re d u ct io n ( % ) Concentration of samples (%, v/v, Test sample/Sterile media) Porphyromonas gingivalis Biofilm Reduction by L. rhamnosus ATCC 7469 CFS L. rhamnosus ATCC 7469 Chlorhexidine Untreated 41 4.3 Transcriptomic Analysis of P. gingivalis 4.3.1 Mapping Analysis of Treated and Untreated P. gingivalis. The mapping analysis was carried with P. gingivalis ATCC 33277 as the reference genome. The total mapping rates (Table 4.3) were above 99% for both treated and untreated P. gingivalis. The preferred multiple mapping ratesare below than 10% and both samples reported less than 5% multiple mapping rate. Table 4.3: The mapping results for treated and untreated P. gingivalis. Sample name Treated P. gingivalis Untreated P. gingivalis Total reads 17671550 18353180 Total mapped reads 17581281 18266026 Uniquely mapped reads 16771614 17743343 Multiple mapped reads 809667 522683 Total mapping rate 99.49% 99.53% Uniquely mapping rate 94.91% 96.68% Multiple mapping rate 4.58% 2.85% 42 4.3.2 Gene Expression Level The gene expressions level of P. gingivalis was measured by transcript abundancy based on the gene length and sequencing depth. The analysis on P. gingivalis RNA showed that L. rhamnosus ATCC 7469 CFS had a siginifcant effect on the gene expression of P. gingivalis compared to the untreated P. gingivalis. As shown in Figure 4.4, out of 1848 genes, a total of 327 genes were expressed differentially. The differential gene expressions include 188 upregulated genes and 139 downregulated genes. Figure 4.4: The comparison of upregulated and downregulated genes between the treated and untreated group of P. gingivalis. 327 188 139 0 50 100 150 200 250 300 350 All Upregulated Downregulated D EG c o u n t Treated vs Untreated Gene Expression Level 43 The gene expression changes are as shown in the heatmap (Figure 4.5) where it showed genes with high fold change. The highly expressed genes are marked in red while the lowly marked genes are marked in blue or grey. The columns represented the two groups of samples, treated and untreated while each row the genes that were differentially expressed. Figure 4.5: The heatmap of the differentially expressed genes. 44 Other than heatmap, the volcano plot is also important to infer the overall statistical distribution of the differentially expressed genes. As shown in Figure 4.6, the upregulated genes were marked in red while the downregulated genes were marked in green. The similarly expressed genes were marked in blue.The horizontal axis indicated the fold change of the genes while the vertical axis indicated the statistical significance of the gene expression. The smaller log p-value indicated higher statistical significance of the gene expressed. Figure 4.6: The volcano plot for all expressed genes. 45 4.3.3 Differentially Expressed Genes in Treated P. gingivalis The treated group of P. gingivalis showed differential expression of some genes when being compared with the untreated group. Several genes related to DNA regulatory proteins, cellular metabolism, bacterial signalling, and virulent factors were significantly downregulated as shown in Table 4.4. Genes such as dnaK, dnaJ, and polA are regulatory and chaperone genes involved in the molecular activity of P. gingivalis. Meanwhile, genes like pdxH, galE, ablA, kdsA, purH, hflX, folD, nifJ, mgtE, pdxB, purD, and rfbA are involved in cellular metabolic activity especially in the glycolysis and electron transport chain. Additionally, genes like mfa1, mfa3, and mfa4 are genes involved in fimbrial activity which is one of the virulent factors of P. gingivalis. The gene ftsY is the only geneinvolved in cellular signal recognition. 46 Table 4.4: The significantly downregulated genes in the treated group of P. gingivalis. Gene name Log2 fold change P-value Gene description dnaK -1.8349 1.54E-05 Molecular chaperone DnaK dnaJ -1.7222 5.74E-05 Molecular chaperone DnaJ galE -1.7101 5.74E-05 NAD dependent epimerase/dehydratase family ablA -1.6468 9.98E-05 Lysine-2,3-aminomutase family kdsA -1.6250 0.000137 3-deoxy-8-phosphooctulonate synthase purH -1.5605 0.000205 Bifunctional phosphoribosylaminoimidazolecarboxamide formyltransferase hflX -1.5534 0.000243 50S ribosome-binding GTPase pdxH -2.0208 0.000403 Pyridoxamine 5'-phosphate oxidase folD -1.4239 0.000979 Tetrahydrofolate dehydrogenase/cyclohydrolase, NAD(P)-binding domain nifJ -1.3233 0.001570 Pyruvate:ferredoxin (flavodoxin) oxidoreductase mfa1 -1.3333 0.001655 Minor fimbrial subunit Mfa1 mfa4 -1.3461 0.00167 Minor fimbrial subunit Mfa4 nrfA -1.2660 0.002477 Ammonia-forming cytochrome C nitrite reductase mgtE -1.2717 0.002525 Cation (magnesium) transporter pdxB -1.2076 0.004307 D-isomer specific 2-hydroxyacid dehydrogenase, NAD binding domain purD -1.1343 0.007428 Phosphoribosylglycinamide synthetase, ATP- grasp (A) domain mfa3 -1.1298 0.011486 Fimbrial tip subunit Mfa3 polA -1.0701 0.011855 DNA polymerase I ftsY -1.0367 0.017097 Signal recognition particle-docking protein 47 On the other hand, genes related to ribosomal subunits, protein synthesis, protein folding, protein assembly, and some virulent genes were significantly upregulated in the treated P. gingivalismolecular expression. Table 4.5 shows the genes that are significantly upregulated. As shown in the table, a lot of ribosomal protein genes were upregulated which includes 50S and 30S ribosomal proteins. The genes are rplQ, rpsD,rpmA, rplU, rpsN, rpsO, rpsM, rplC, rplS, rpsG, and rplX. Other than that, there are a few metabolism related genes such as gpmA, nadA, miaA, panC, coaE, and serC. Additionally, a few assembly genes were also upregulated in the treated group of P. gingivalis including rimP and bamD. Other upregulated genes belong to virulent genes such as rgpB and rgpA, resolvase gene ruvX and heme chaperone gene hemW. Table 4.5: The significantly upregulated genes in the treated group of P. gingivalis. Gene name Log2 fold change P-value Gene description ruvX 2.4745 8.59E-08 Holliday junction resolvase gpmA 2.5345 8.00E-06 Putative 2,3-bisphosphoglycerate-dependent phosphoglycerate rplQ 1.7843 3.44E-05 50S ribosomal protein L17 rpsD 1.7438 0.000144 30S ribosomal protein S4 nadA 2.3011 0.000283 Quinolinate synthase rpmA 1.5461 0.000309 50S ribosomal protein L27 rplU 1.5543 0.000354 50S ribosomal protein L21 rpsN 1.4569 0.000611 30S ribosomal protein S14 rpsO 1.4881 0.000662 30S ribosomal protein S15 rgpB 1.3235 0.001720 Arg-gingipain RgpB 48 rgpA 1.2903 0.002164 Arg-gingipain RgpA rpsM 1.3617 0.003352 30S ribosomal protein S13 rimP 1.3008 0.003534 Ribosome assembly cofactor RimP miaA 1.4931 0.006048 tRNA (adenosine(37)-N6)- dimethylallyltransferase rplC 1.2019 0.006903 50S ribosomal protein L3 rplS 1.1823 0.006995 50S ribosomal protein L19 bamD 1.4487 0.007262 Outer membrane protein assembly factor panC 1.4722 0.011442 Pantoate-beta-alanine ligase rpsJ 1.1781 0.011557 30S ribosomal protein S10 coaE 1.8469 0.016051 Dephospho-CoA kinase serC 1.0163 0.024314 Putative phosphoserine transaminase rpsG 1.1059 0.024467 30S ribosomal protein S7 hemW 2.9271 0.027042 Radical SAM family heme chaperone rplX 1.1422 0.039811 50S ribosomal protein L24 49 4.3.4 Gene Ontology (GO) Enrichment Analysis Investigation of the gene ontology (GO) of the treated P. gingivalis were also done to investigate the affected biological functions and metabolic pathway. The GO results for all differentially expressed genes are as shown in Figure 4.7. The GO of downregulated genes are as shown in Figure 4.8 while the GO of upregulated genes are as shown in Figure 4.9. The GO analysis is divided into three groups of biological functions which are molecular function (MF), cellular components (CC), and biological process (BP). The significant GO are marked with asterisk. (a) 50 (b) (c) Figure 4.7: Overall gene ontology analysis (a) Gene ontology analysis of all differentially expressed genes in the MF category where the highest differentially expressed genes were in the ligase forming activity and endopeptidase activity group. (b) Gene ontology in the cellular components showed the differential expression of respective activity as shown in the figure. (c) Gene ontology in binding process category where all genes were expressed similarly in all categories. 51 (a) (b) 52 (c) Figure 4.8: Downregulated gene oncology analysis (a) Gene ontology analysis of downregulated genes in the MF category showed the genes were downregulated in similar amounts for all MF activities. (b) Gene ontology analysis of downregulated genes in the CC category where genes under membrane components were slightly downregulated. (c) Gene ontology in BP category where all genes were similarly downregulated in all activities. 53 (a) (b) 54 (c) Figure 4.9: Upregulated gene oncology analysis (a) Gene ontology analysis of upregulated genes in the MF category showed that genes in catalytic activity and peptidase activity were the highest upregulated genes. (b) Gene ontology analysis of upregulated genes in the CC category where protein-containing complex were the highest upregulated activity. (c) Gene ontology in BP category showed that genes under proteolysis activity and preotein metabolic process were the highly upregulated genes in the BP category. 55 4.3.5 KEGG Enrichment Analysis The connection of several genes might be integrated together in a biological function and it is studied in an analysis called KEGG enrichment analysis. KEGG is a database for genomes, biological pathways, disease pathways, drugs metabolism, and chemical reactions. KEGG is applied in bioinformatics to understand the genomics, transcriptomics, metabolomics, and a few other branches of biological study. This study utilized KEGG analysis to understand the significantly enriched pathways associated with the differentially expressed genes. The scatter plots for KEGG enrichment are as shown in Figure 4.10. As shown in Figure 4.10 (a) the genes that are highly downregulated belongs to the two-component system, quorum sensing, purine metabolism, and microbial metabolism in diverse environment groups. Meanwhile, in the upregulated KEGG pathways shown in Figure 4.10 (b), the significantly upregulated genes belong to ribosome pathway, oxidative phosphorylation, glycine, serine, and threonine metabolism and biosynthesis of amino acids pathways. The detailed information of each pathway is as shown in Table 4.6. 56 (a) (b) Figure 4.10: KEGG enrichment analysis (a) the distribution of downregulated genes involved in KEGG pathways. (b) The distribution of upregulated genes involved in stated KEGG pathways. Gene count indicated the number of differentially expressed genes under the pathways, gene ratio indicated the ratio of gene counts in the pathway over overall genes while multi-coloured padj represents the adjusted p-value for each pathways. 57 Table 4.6: Detailed information on each affected KEGG pathways, significantly affected pathways are marked with asterisk. KEGG Pathway ID Pathway Description Related Genes ID Differentially Expressed Genes in Pathways pgn02024 Quorum sensing (downregulated) PGN_1733, PGN_1733, PGN_0599, PGN_0264. ftsY pgn02020 Two-component system (downregulated) PGN_0715, PGN_1162, PGN_1041. - pgn00230 Purine metabolism (downregulated) PGN_0865, PGN_1948, PGN_1148, PGN_1396. purH, purD pgn01120 Microbial metabolism in diverse environments (downregulated) PGN_0403, :PGN_1206, PGN_1418, PGN_0351, PGN_1746, PGN_1162. gpmA, pdxH, folD, nifJ, nrfA, serC pgn03010* Ribosome (upregulated) PGN_1840, PGN_1842, PGN_1647, PGN_1648, PGN_1855, PGN_1698, PGN_1844, PGN_0167, PGN_1868, PGN_0035, PGN_0279, PGN_1869, PGN_1855, PGN_1871, PGN_1857. rplQ, rpsD, rpmA, rplU, rpsN, rpsO, rpsM, rplC, rplS, rpsJ pgn00190* Oxidative phosphorylation (upregulated) PGN_1761, PGN_1758, PGN_1762, PGN_1761. - pgn00260 Glycine, serine and threonine metabolism (upregulated) PGN_0243, PGN_0611, PGN_1489, PGN_1495, PGN_0612. gpmA, serC 58 pgn00260 Biosynthesis of amino acids (upregulated) PGN_0243, PGN_1080, PGN_0351, PGN_0611, PGN_0351, PGN_1874, PGN_1996, PGN_1495, PGN_0612, PGN_1234. gpmA, serC 59 4.3.6 Validation of Gene of Interest by Real-Time Quantitative PCR (RT - QPCR) The genes of interest detected in the NGS procedure are validated using RT - qPCR technique. The gene of interest includes fimA, mfa1, kgp, and rgp with 16s rRNA acted as a reference gene. The concentration and purity of the extracted RNA were determined before RT - qPCR procedure. The purity of RNA was determined based on the ratio of absorbance at 260nm to absorbance at 280nm (A260/A280). The concentration of RNA from untreated sample was 1206.40 ng/µl with the purity of 2.038 (Table 4.5). Meanwhile, the RNA concentration of P. gingivalis treated with L. rhamnosus ATCC 7469 CFS was 1776.24 ng/µl with the purity of 2.026. The quality of RNA was also assessed by gel electrophoresis method (Figure 4.9). The extracted RNA was observed to be intact and pure with the presence of singular intense band for each sample. The treated and untreated RNA were then converted into cDNA to be used as template for RT-qPCR. Table 4.7: The quantification of RNA concentration and RNA purity determined by molecular spectrophotometer. Samples RNA concentration (ng/µl) RNA purity (A260:A280) Untreated P. gingivalisRNA 1206.40 2.038 Treated P. gingivalisRNA 1776.24 2.026 60 Figure 4.11: The visualization of gel electrophoresis to assess the quality of extracted RNA (A: 1000bp DNA ladder, B1: Untreated P.gingivalis RNA sample 1, B2: Untreated P.gingivalis RNA sample 2, C1: Treated P.gingivalis RNA sample 1, C2: Treated P.gingivalis RNA sample 2). Then, the detected Ct value from the qPCR procedures were translated as fold changes by the following calculations: Fold change=(∆CTD-∆CTB)-(∆CTC-∆CTA) ΔCTD represents reference gene 16s rRNA from untreated samples, ΔCTB represents reference gene 16s rRNA from sample treated with L. rhamnosus ATCC 7469 CFS, ΔCTC refers to the gene of interest from treated samples, ΔCTA refers to the gene of interest from untreated sample. 61 The relative expression is the fold change of target genes in P. gingivalis treated with L. rhamnosus ATCC 7469 CFS relative to untreated P. gingivalis sample with the normalization to the reference gene, 16s rRNA. The relative expression of selected genes in treated samples, fimA, mfa1, kgp, and rgp were recorded as 3.77± 1.78, 1.66± 2.00, - 1.46± 4.07, and -1.01± 3.79 respectively (Figure 4.10) where mfa1 gene was downregulated while fimA, kgp, and rgpgenes were upregulated. Figure 4.12: The relative expression of the selected genes in P. gingivalis treated with L. rhamnosus ATCC 7469 CFS. -6.00 -4.00 -2.00 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 16s rRNA fimA mfa1 kgp rgp R e la ti ve e xp re ss io n ( fo ld c h an ge s) Relative Expression of Selected Genes