Introduction
Materials and methods
Bacterial strains
Target gene
| Sequence (5’-3’) | aT (°C) | Amplified fragment | Amplicon size (bp) | Reference |
---|---|---|---|---|---|
Sa442
| 5’-AATCTTTGTCGGTACACGATATTCTTCACG-3’ 5’-CGTAATGAGATTTCAGTAAATACAACA-3’ | 55 | Sa442 protein | 108 | [7] |
mecA
| 5’-ACCAGATTACAACTTCACCAGG-3’ 5’-CCACTTCATATCTTGTAACG-3’ | 45 | Penicillin binding protein 2a | 162 | [7] |
icaA | 5’-ACACTTGCTGGCGCAGTCAA-3’ 5’-TCTGGAACCAACATCCAACA-3’ | 55 | Intercellular adhesion A | 188 | [24] |
icaD | 5’-ATGGTCAAGCCCAGACAGAG-3’ 5’-AGTATTTTCAATGTTTAAAGCAA-3’ | 55 | Intercellular adhesion D | 198 | [24] |
Can
| 5’-GTCAAGCAGTTATTAACACCAGAC-3’ 5’-AATCAGTAATTGCACTTTGTCCACTG-3’ | 62 | Collagen adhesion | 192 | [36] |
fnbA | 5’-CATAAATTGGGAGCAGCATCA-3’ 5’-ATCAGCAGCTGAATTCCCATT-3’ | 62 | Fibronectin binding protein A | 191 | [36] |
fnbB | 5’-GTAACAGCTAATGGTCGAATTGATACT-3’ 5’-CAAGTTCGATAGGAGTACTATGTTC-3’ | 62 | Fibronectin binding protein B | 201 | [36] |
sspA
| 5’-GACAACAGCGACACTTGT 3’ 3’-AGTATCTTTACCTACAACTACA-5’ | 45 | Serine protease | 292 | [40] |
sspB
| 5’-TGAAGAAGATGGCAAAGTTAG-3’ 3’-TTGAGATACACTTTGTGCAAG-5’ | 47 | Cysteine protease | 493 | [40] |
Geh
| 5’-GCACAAGCCTCGG -3’ 3’-GACGGGGGTGTAG-5’ | 40 | Lipase | 473 | [45] |
Characterization of the enzymatic activity
Phenotypic determination of slime production
Semi-quantitative adherence assay
Detection of icaA, icaD, fnbA, fnbB, cna, sspA, sspB and geh genes
Statistical analysis
Results
Distribution of isolates
Characterization of the enzymatic activity
Strains | Exoenzymes expression | Hemolysis type | |||
---|---|---|---|---|---|
Lecithinase | Lipase | Caseinase | Gelatinase | ||
ATCC6538 | + | + | + | + | Beta |
1 | + | + | + | + | Alpha |
2 | + | + | + | + | Alpha |
3 | + | + | + | + | Alpha |
4 | + | + | + | + | Alpha |
5 | + | + | + | + | Beta |
6 | + | + | + | + | Beta |
7 | + | + | + | + | Beta |
8 | - | - | + | + | Beta |
9 | + | + | + | + | Alpha |
10 | + | + | + | + | Beta |
11 | + | + | + | + | Alpha |
12 | + | + | + | + | Beta |
13 | + | + | + | + | Beta |
14 | + | - | + | + | Alpha |
15 | + | + | + | + | Alpha |
16 | + | - | + | + | Beta |
17 | + | - | + | + | Beta |
18 | + | + | + | + | Alpha |
19 | + | + | + | + | Beta |
20 | + | + | + | + | Alpha |
21 | + | + | + | + | Beta |
22 | + | + | + | + | Alpha |
23 | - | - | + | + | Beta |
24 | + | + | + | + | Alpha |
25 | + | + | + | + | Alpha |
26 | + | + | + | + | Beta |
27 | + | + | + | + | Beta |
28 | + | + | + | + | Alpha |
29 | + | + | + | + | Alpha |
30 | - | - | + | + | Beta |
31 | + | + | + | + | Beta |
32 | + | + | + | + | Alpha |
33 | + | + | + | + | Alpha |
34 | + | + | + | + | Alpha |
35 | + | + | + | + | Beta |
36 | + | - | + | + | Alpha |
37 | + | + | + | + | Beta |
38 | + | + | + | + | Beta |
39 | + | + | + | + | Alpha |
40 | + | - | + | + | Beta |
41 | + | + | + | + | Alpha |
42 | + | + | + | + | Beta |
43 | + | - | + | + | Beta |
% expression | 93,0% | 79,1% | 100% | 100% | 48.9%(alpha);51.1%(beta) |
Phenotypic determination of slime production
Strains | Biofilm phenotype (CRA) | OD570 ± SD | Adherence state |
---|---|---|---|
ATCC 6538 | S+ | 2.90±0.05 | highly positive |
ATCC 43300 | S+ | 0.71±0.15 | highly positive |
1 | S- | 0.19±0.05 | moderately positive |
2 | S- | 0.12±0.03 | moderately positive |
3 | S+ | 0.13±0.02 | moderately positive |
4 | S+ | 0.59±0.11 | highly positive |
5 | S+ | 0.46±0.02 | highly positive |
6 | S+ | 0.26±0.01 | highly positive |
7 | S+ | 0.22±0.08 | moderately positive |
8 | S+ | 0.42±0.01 | highly positive |
9 | S- | 0.15±0.02 | moderately positive |
10 | S+ | 0.80±0.03 | highly positive |
11 | S+ | 2.96±0.08 | highly positive |
12 | S- | 0.15±0.04 | moderately positive |
13 | S- | 0.15±0.07 | moderately positive |
14 | S+ | 1.77±0.02 | highly positive |
15 | S+ | 0.22±0.01 | moderately positive |
16 | S+ | 0.17±0.03 | moderately positive |
17 | S+ | 2.62±0.01 | highly positive |
18 | S+ | 0.24±0.02 | highly positive |
19 | S+ | 0.13±0.05 | moderately positive |
20 | S+ | 0.62±0.03 | highly positive |
21 | S+ | 0.12±0.03 | moderately positive |
22 | S+ | 0.21±0.39 | moderately positive |
23 | S+ | 0.14±0.06 | moderately positive |
24 | S+ | 0.14±0.01 | moderately positive |
25 | S+ | 0.14±0.01 | moderately positive |
26 | S+ | 1.00±0.12 | highly positive |
27 | S- | 0.18±0.13 | moderately positive |
28 | S+ | 0.16±0.30 | moderately positive |
29 | S- | 0.26±0.60 | highly positive |
30 | S+ | 0.14±0.02 | moderately positive |
31 | S- | 0.20±0.03 | moderately positive |
32 | S+ | 1.51±0.08 | highly positive |
33 | S+ | 0.41±0.09 | highly positive |
34 | S+ | 0.40±0.07 | highly positive |
35 | S+ | 0.27±0.06 | highly positive |
36 | S+ | 0.31±0.04 | highly positive |
37 | S+ | 0.23±0.02 | highly positive |
38 | S+ | 0.37±0.05 | highly positive |
39 | S- | 0.18±0.02 | moderately positive |
40 | S- | 0.16±0.03 | moderately positive |
41 | S- | 0.22±0.04 | moderately positive |
42 | S- | 0.25±0.05 | highly positive |
43 | S- | 0.12±0.03 | moderately positive |
Semi-quantitative adherence assay
Detection by PCR of icaA, icaD, fnbA, fnbB, cna, sspA, sspB and geh genes
Statistical analysis
Association between Congo red phenotypic test and adherence assay
Association between biofilm production on polystyrene and biofilm related genes
Genes | Moderately bioflm producers (n=23) | Highly biofilm producers (n=20) | P (univariate analysis) | P (multivariate analysis) | |
---|---|---|---|---|---|
MSCRAMMs genes |
fnbA+
| 14/23 | 18/20 | 0,001a | 0,040 |
fnbB+
| 2/23 | 6/20 | 0,002a | 0,003 | |
cna+
| 15/23 | 17/20 | 0,154a | 0,981 | |
Biofilm production control genes |
icaA+
| 13/23 | 17/20 | 0,143a | 0,661 |
icaD+
| 14/23 | 13/20 | 0,546 | - |
Association between biofilm production on polystyrene and exoenzymes genes
Exoenzymes genes | Moderately bioflm producers (n=23) | Highly biofilm producers (n=20) | P (univariate analysis) | P (bivariate analysis) |
---|---|---|---|---|
geh+
| 8/23 | 5/20 | 0,791 | - |
sspA+
| 6/23 | 9/20 | 0,117a | 0,223 |
sspB+
| 1/23 | 5/20 | 0,013a | 0,003 |
Discussion
-
With regard to the complexity and the variability of the biofilm phenotype, genetic studies identified numerous genes involved in biofilm formation. However, their relative importance is still unclear [42]. In the light of this, our multivariate analysis showed that the presence of fnbA and fnbB genes was significantly associated with a high biofilm production on polystyrene. This consolidates the alleged role of these genes in biofilm formation. Comparably, some studies suggested that detection of some adhesion factors is more practical for the prediction of biofilm formation [4, 31, 43]. Other reports have shown the decisive role of the ica gene [32, 33]. However, no statistical difference was found in the distribution of genes by some researchs [28, 31]. Such investigations aim to find new attractive targets for antivirulence therapy by overcoming biofilm formation [31].