Fig. 1
Fig. 2
Relationship between drug resistance phenotype and drug resistance genotype of S_ agalactiae
| Antibiotic type | Resistance gene | Genotype (%) | Phenotype resistance rate (%) | Consistency rate (%) | |
|---|---|---|---|---|---|
| Number of bacteria | Positive rate | ||||
| ermB | 11/15 | 73.33 | 91.66 | ||
| Macrolides | ermA | 10/15 | 66.67 | 80.00 | 83.33 |
| ermC | 5/15 | 33.33 | 41.66 | ||
| Lincosamides | lnuA | 9/15 | 60.00 | 66.67 | 89.99 |
| tetM | 7/15 | 46.67 | 100.00 | ||
| tetK | 6/15 | 40.00 | 85.71 | ||
| Tetracyclines | tetS | 6/15 | 40.00 | 46.67 | 85.71 |
| tetO | 5/15 | 33.33 | 71.42 | ||
The combined virulence gene profile of S_ agalactiae
| Virulence gene profile | Distributiona (%) |
|---|---|
| glnA+hylB+bibA+iagA+fbsA+clyE | 3 (20) |
| glnA+hylB+iagA+fbsA | 2 (13.33) |
| bca+glnA+hylB+iagA+fbsA+clyE | 1 (6.67) |
| bca+cfb+hylB+bibA+iagA+fbsA+clyE | 1 (6.67) |
| bibA+iagA+fbsA+clyE | 1 (6.67) |
| bca+hylB+bibA+iagA+fbsA | 1 (6.67) |
| hylB+bibA+iagA+fbsA | 1 (6.67) |
| bca+bibA+iagA+fbsA | 1 (6.67) |
| hylB+bibA+iagA+fbsA+clyE | 1 (6.67) |
| cfb+hylB+bibA+iagA+fbsA | 1 (6.67) |
| cfb+hylB+bibA+fbsA+clyE | 1 (6.67) |
Primer sequence and reaction conditions of the drug resistance gene of S_ agalactiae
| Antimicrobial drug class | Resistance gene | Primer sequence (5′–3′) | Annealing temperature (°C) | PCR product size (bp) | Reference |
|---|---|---|---|---|---|
| F: GGTGGCTGGGGGGTAGATGTATTAACTGG | |||||
| Lincosamides | lnuA | R: GCTCTCTTTGAAATACATGGTATTTTTCGATC | 56 | 323 | (23) |
| F: AACTTAGGCATTCTGGCTCAC | |||||
| tetO | R: TCCCACTGTTCCATATCGTCA | 50 | 515 | (29) | |
| F: TCGATAGGAACAGCAGTA | |||||
| tetK | R: CAGCAGATCCTACTCCTT | 44 | 169 | (33) | |
| Tetracyclines | |||||
| tetM | F: GTGGACAAAGGTACAACGAG | 50 | 406 | ||
| R: CGGTAAAGTTCGTCACACAC | |||||
| (39) | |||||
| F: CATAGACAAGCCGTTGACC | |||||
| tetS | R: ATGTTTTTGGAACGCCAGAG | 48 | 667 | ||
| ermB | F: CGAGTGAAAAAGTACTCAACC | 48 | 652 | ||
| R: AGTAACGGTACTTAAATTGTTTAC | |||||
| F: ATCTTTGAAATCGGCTCAGG | |||||
| Macrolides | ermC | R: CAAACCCGTATTCCACGATT | 47 | 295 | (32) |
| F: GTTCAAGAACAATCAATACAGAG | |||||
| ermA | R: GGATCAGGAAAAGGACATTTTAC | 48 | 421 |
Minimum inhibitory concentration (MIC) values of, and resistance of S_ agalactiae isolates to, β-lactams, sulfonamides and tetracyclines
| Isolate no. | MIC (μg/mL) | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| PEN | A/C | AMP | CET | OXA | SMZ | SXT | TE | DOX | |
| SH07 | ≤0.125 (S3) | 1/0.5 (R) | ≤0.125 (S) | 32 (R) | 1 (S) | 4 (S) | 0.5/9.5 (S) | 4 (S) | 4 (S) |
| SH07-2 | 4 (R) | ≤0.25/0.12 (S) | ≤0.125 (S) | 0.5 (S) | 16 (R) | 256 (I) | 0.25/4.8 (S) | 16 (R) | 2 (S) |
| SH12 | ≤0.125 (S) | ≤0.25/0.12 (S) | 0.25 (I) | 0.5 (S) | 4 (R) | 8 (S) | 0.5/9.5 (S) | 32 (R) | 4 (S) |
| SH33 | ≤0.125 (S) | ≤0.25/0.12 (S) | ≤0.125 (S) | 0.5 (S) | 16 (R) | 64 (S) | 0.5/9.5 (S) | 32 (R) | 0.5 (S) |
| SH45 | ≤0.125 (S) | ≤0.25/0.12 (S) | ≤0.125 (S) | 1 (S) | 32 (R) | 64 (S) | 0.25/4.8 (S) | 64 (R) | 2 (S) |
| HLJ008 | ≤0.125 (S) | ≤0.25/0.12 (S) | ≤0.125 (S) | 32 (R) | 32 (R) | 128 (S) | 0.25/4.8 (S) | 32 (R) | 2 (S) |
| HLJ048-2 | 2 (R) | ≤0.25/0.12 (S) | ≤0.125 (S) | 1 (S) | 4 (R) | 2 (S) | 0.5/9.5 (S) | 16 (R) | 8 (I) |
| HLJ030-3 | ≤0.125 (S) | ≤0.25/0.12 (S) | ≤0.125 (S) | 0.5 (S) | 0.5 (S) | 64 (S) | 0.5/9.5 (S) | 64 (R) | 4 (S) |
| NM025 | ≤0.125 (S) | ≤0.25/0.12 (S) | ≤0.125 (S) | 1 (S) | 1 (S) | 64 (S) | 0.25/4.8 (S) | 32 (R) | 16 (R) |
| HB016 | ≤0.125 (S) | ≤0.25/0.12 (S) | ≤0.125 (S) | 0.25 (S) | 16 (R) | 128 (S) | 0.5/9.5 (S) | 16 (R) | 4 (S) |
| NM-2-72-4 | 8 (R) | ≤0.25/0.12 (S) | 2 (R) | 1 (S) | 16 (R) | 128 (S) | 0.25/4.8 (S) | 32 (R) | 32 (R) |
| NM-2-034-3 | 8 (R) | 16/8 (R) | 4 (R) | 0.5 (S) | 4 (R) | 64 (S) | 2/38 (I) | 4 (S) | 4 (S) |
| SH-2-46-1 | ≤0.125 (S) | ≤0.25/0.12 (S) | ≤0.125 (S) | 0.5 (S) | 32 (R) | 128 (S) | 0.25/4.8 (S) | 16 (R) | 4 (S) |
| SH-2-14-2 | 2 (R) | 4/2 (R) | ≤0.125 (S) | 1 (S) | 16 (R) | 128 (S) | 0.25/4.8 (S) | 32 (R) | 8 (I) |
| SD-2-009-2 | ≤0.125 (S) | 16/8 (R) | ≤0.125 (S) | 0.5 (S) | 4 (R) | 128 (S) | 0.25/4.8 (S) | 8 (I) | 4 (S) |
| Drug resistance rate (%) (/15) | |||||||||
| S | 66.67 (10) | 73.33 (11) | 80.00 (12) | 86.67 (13) | 20.00 (3) | 93.33 (14) | 93.33 (14) | 13.33 (2) | 73.33 (11) |
| I | 0 (0) | 0 (0) | 6.67 (1) | 0 (0) | 0 (0) | 6.67 (1) | 6.67 (1) | 6.67 (1) | 13.33 (2) |
| R | 33.33 (5) | 26.67 (4) | 13.33 (2) | 13.33 (2) | 80.00 (12) | 0 (0) | 0 (0) | 80.00 (12) | 13.33 (2) |
Logistic regression analysis of the relationship between antibiotics and virulence genes
| Virulence gene | Penicillin | Cefoxitin | Oxacillin | Doxycycline | Gentamicin | Clindamycin | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| B | Exp(B) | P | B | Exp(B) | P | B | Exp(B) | P | B | Exp(B) | P | B | Exp(B) | P | B | Exp(B) | P | |
| bca | 40.537 | 4.028 | 0.999 | 1.099 | 3.000 | 1.000 | −42.406 | 0.000 | 0.999 | 1.099 | 3.000 | 1.000 | −41.307 | 0.000 | 0.999 | −61.799 | 0.000 | 0.999 |
| scpB | −20.269 | 0.000 | 1.000 | −60.718 | 0.000 | 0.999 | 62.916 | 2.108 | 0.999 | 19.699 | 3.590 | 1.000 | 62.105 | 9.369 | 0.999 | −62.493 | 0.000 | 0.999 |
| glnA | 0.000 | 1.000 | 1.000 | −19.411 | 0.000 | 0.999 | −21.896 | 0.000 | 0.999 | −19.411 | 0.000 | 0.999 | −19.411 | 0.000 | 0.999 | −0.693 | 0.500 | 0.744 |
| cfb | −0.934 | 0.393 | 1.000 | 42.406 | 2.610 | 0.999 | −84.812 | 0.000 | 0.999 | −38.011 | 0.000 | 1.000 | −80.417 | 0.000 | 0.999 | −81.880 | 0.000 | 0.999 |
| hylB | 42.406 | 2.610 | 0.999 | −40.209 | 0.000 | 0.999 | 42.406 | 2.610 | 0.999 | 40.209 | 2.900 | 0.999 | 40.209 | 2.900 | 0.999 | 0.000 | 1.000 | 1.000 |
| bibA | 21.203 | 1.615 | 1.000 | −19.411 | 0.000 | 1.000 | −21.896 | 0.000 | 1.000 | −19.411 | 0.000 | 1.000 | −19.411 | 0.000 | 1.000 | −42.412 | 0.000 | 0.999 |
| iagA | −41.472 | 0.000 | 1.000 | −1.099 | 0.333 | 1.000 | 42.406 | 2.610 | 1.000 | −1.099 | 0.333 | 1.000 | 41.307 | 8.699 | 1.000 | −20.080 | 0.000 | 1.000 |
| cylE | 0.000 | 1.000 | 1.000 | −1.099 | 0.333 | 1.000 | 42.406 | 2.610 | 0.999 | −1.099 | 0.333 | 1.000 | −1.099 | 0.333 | 1.000 | 21.203 | 1.615 | 1.000 |
16 kinds of antibacterial drugs and their classification
| Drug category | Antibacterial drugs |
|---|---|
| penicillin | |
| ampicillin | |
| β-lactams | amoxicillin / clavulanic acid |
| oxacillin | |
| cefoxitin | |
| Macrolides | erythromycin |
| Lincosamides | clindamycin |
| Aminoglycosides | gentamicin |
| Tetracyclines | doxycycline tetracycline |
| Chloramphenicols | florfenicol |
| Ansamycins | rifampicin |
| Glycopeptides | vancomycin |
| Quinolones | ciprofloxacin |
| Sulfonamides | sulfisoxazole |
| sulfamethoxazole |
The drug resistance patterns and gene expression profiles of S_ agalactiae
| Isolate no. | Drug resistance pattern | Resistant gene | Virulence gene |
|---|---|---|---|
| SH07 | (A/C)/CET/ERY | ermB+lnuA+tetM+tetO | bca+hylB+bibA+iagA+fbsA |
| SH07-2 | PEN/OXA/TE/ERY/CLI/FFC | ermA+ermB+lnuA+tetM+tetK+tetS+tetO | glnA+hylB+iagA+fbsA |
| SH12 | OXA/TE/ERY/CLI/GM | ermA+ermB+ermC+lnuA+tetS+tetO | bca+glnA+hylB+iagA+fbsA+clyE |
| SH33 | OXA/TE/ERY/CLI/GM | ermA+ermB+ermC+lnuA+tetK+tetS+tetO | bca+cfb+hylB+bibA+iagA+fbsA+clyE |
| SH45 | OXA/TE/ERY/CLI | ermA+ermB+lnuA+tetK+tetS | hylB+bibA+iagA+fbsA |
| HLJ008 | CET/OXA/TE/ERY/CLI | ermB+ermA+ermC+lnuA+tetM+tetK | glnA+hylB+bibA+iagA+fbsA+clyE |
| HLJ030-3 | TE/CLI/FFC | ermB+ermA+lnuA+tetM | hylB+bibA+iagA+ fbsA+clyE |
| NM025 | TE/DOX/ERY | ermA+ermB+lnuA+tetK+tetO | glnA+hylB+bibA+iagA+fbsA+clyE |
| HB016 | OXA/TE/ERY/CLI/GM | ermA+ermB+ermC+lnuA+tetM+tetK+tetS | glnA+hylB+bibA+iagA+fbsA+clyE |
| SH-2-46-1 | OXA/TE/ERY | ermA+ermB+lnuA+tetO | glnA+hylB+iagA+fbsA |
Primer sequence and reaction conditions of the virulence gene of S_ agalactiae (13, 22, 23)
| Virulence factor | Virulence gene | Primer sequence (5′–3′) | Annealing temperature (°C) | PCR size product (bp) |
|---|---|---|---|---|
| F: CATTGCGTAGTCACCTCCC | ||||
| β-haemolysin/cytolysis | cylE | R: GGGTTTCCACAGTTGCTTGA | 54 | 399 |
| F: TAACAGTTATGATACTTCACAGAC ⎕ | ||||
| αC protein | Bca | R: ACGACTTTCTTCCGTCCACTTAGG | 51 | 535 |
| F: CCAAGACTTCAGCCACAAGG | ||||
| C5a peptidase | scpB | R: CAATTCCAGCCAATAGCAGC | 57 | 591 |
| F: ACCGTCTGAAATGATGTGG | ||||
| Laminin binding protein | lmb | R: GATTGACGTTGTCTTCTGC | 51 | 572 |
| Glutamine synthetase | glnA | F: ACGTATGAACAGAGTTGGCTATAA | 52 | 471 |
| R: TCCTCTGATAATTGCATTCCAC | ||||
| CAMP factor | Cfb | F: ATGGGATTTGGGATAACTAAGCTAG | 52 | 193 |
| R: AGCGTGTATTCCAGATTTCCTTAT | ||||
| Hyaluronidase | hylB | F: ACAAATGGAACGACGTGACTAT | 52 | 346 |
| R: CACCAATTGGCAGAGCCT | ||||
| F: AAGCAACTAGAAGAGGAAGC | ||||
| βC protein | bac | R: TTCTGCTCTGGTGTTTTAGG | 53 | 479 |
| Bacterial immunogenic | bibA | F: AACCAGAAGCCAAGCCAGCAACC | 58 | 127 |
| adhesive | R: AGTGGACTTGCGGCTTCACCC | |||
| F: CGGGATTGATCTAAGTCGCT | ||||
| Invasion-associated gene | iagA | R: CCATCAACATCAGTCGCTAA | 53 | 459 |
| F: AGAGCCAAGTAGGTCAACTTATAG | ||||
| Fibrin binding protein B | fbsA | R: TTCATTGCGTCTCAAACCG | 54 | 290 |
Minimum inhibitory concentration (MIC) values of, and resistance of S_ agalactiae isolates to, a macrolide, a lincosamide, an aminoglycoside, a quinolone, a chloramphenicol, an ansamycin, and a glycopeptide
| Isolate no. | MIC (μg/mL) | ||||||
|---|---|---|---|---|---|---|---|
| ERY | CLI | GM | CIP | FFC | RIF | VAN | |
| SH07 | 16 (R) | 0.25 (S) | 4 (S) | 0.5 (S) | 2 (S) | 0.5 (S) | 0.5 (S) |
| SH07-2 | 16 (R) | 4 (R) | 4 (S) | 0.5 (S) | 16 (R) | 0.125 (S) | 0.5 (S) |
| SH12 | 32 (R) | 8 (R) | 16 (R) | 0.5 (S) | 4 (S) | 0.5 (S) | 0.5 (S) |
| SH33 | 16 (R) | 8 (R) | 16 (R) | 0.5 (S) | 8 (I) | 0.5 (S) | 0.5 (S) |
| SH45 | 64 (R) | 8 (R) | 2 (S) | 2 (I) | 8 (I) | 0.5 (S) | 0.5 (S) |
| HLJ008 | 16 (R) | 4 (R) | 4 (S) | 0.5 (S) | 4 (S) | 0.5 (S) | 0.5 (S) |
| HLJ048-2 | 16 (R) | 16 (R) | 8 (I) | 0.5 (S) | 4 (S) | 1 (S) | 0.5 (S) |
| HLJ030-3 | 0.5 (S) | 16 (R) | 2 (S) | 0.5 (S) | 16 (R) | 0.5 (S) | 0.5 (S) |
| NM025 | 16 (R) | 0.5 (S) | 1 (S) | 0.5 (S) | 8 (I) | 0.5 (S) | 0.5 (S) |
| HB016 | 16 (R) | 4 (R) | 16 (R) | 0.5 (S) | 4 (S) | 0.5 (S) | 0.5 (S) |
| NM-2-72-4 | 2 (I) | 0.5 (S) | 32 (R) | 0.5 (S) | 16 (R) | 0.5 (S) | 0.5 (S) |
| NM-2-034-3 | 0.5 (S) | 16 (R) | 4 (S) | 4 (R) | 16 (R) | 0.5 (S) | 0.5 (S) |
| SH-2-46-1 | 16 (R) | 0.5 (S) | 4 (S) | 0.5 (S) | 4 (S) | 0.5 (S) | 0.5 (S) |
| SH-2-14-2 | 16 (R) | 16 (R) | 4 (S) | 4 (R) | 8 (I) | 0.5 (S) | 0.5 (S) |
| SD-2-009-2 | 32 (R) | 0.5 (S) | 16 (R) | 4 (R) | 16 (R) | 0.5 (S) | 0.5 (S) |
| Drug resistance rate (%) (/15) | |||||||
| S | 13.33 (2) | 33.33 (5) | 60.00 (9) | 73.33 (11) | 40.00 (6) | 100 (15) | 100 (15) |
| I | 6.67 (1) | 0 (0) | 6.67 (1) | 6.67 (1) | 26.67 (4) | 0 (0) | 0 (0) |
| R | 80.00 (12) | 66.67 (10) | 33.33 (5) | 20.00 (3) | 33.33 (5) | 0 (0) | 0 (0) |