Abstract
This study aimed to evaluate the activity, resistance, clonality of MIC distribution, and the correlation between virulence and resistance genes and biofilm formation of omadacycline (OMC) in clinics for Streptococcus agalactiae isolates from China. 162 isolates were collected retrospectively in China. The S. agalactiae were collected from the body's cervical secretions, wound secretions, ear swabs, secretions, semen, venous blood, cerebrospinal fluid, pee, etc. The MIC of OMC against S. agalactiae was determined by broth microdilution. The inhibition zone diameters of OMC and other common antibiotics were measured using filter paper. D-test was performed to determine the phenotype of cross resistance between erythromycin and clindamycin. In Multilocus sequence typing (MLST), some commonly-detected resistance genes and virulence gene of these S. agalactiae isolates were investigated using polymerase chain reaction (PCR). Biofilms were detected by crystal violet staining. Our data demonstrated the correalation of the biofilm formation and OMA antimicrobial susceptibility of S.agalactiae clinical isolates with the carrier of virulence gene scpB. Conclusively, OMC exhibits the robust antimcirobial activity against clinical S. agalactiae isolates from China compared with DOX or MIN, and the carrier of the virulence gene scpB might correlate with the biofilm formation in OMC-resistant S. agalactiae.
1. Introduction
S. agalactiae belongs to group B Streptococcus (GBS) [1, 2]. S. agalactiae is a gram-positive cocci that is microscopically examined for long-chain or long-chain chain arrangement. There are 10 serotypes in III, IV, V, VI, VII, VIII, and IX [3, 4]. S. agalactiae is ubiquitous in the natural environment and can reside in the human reproductive tract and digestive tract [5], which is the main pathogenic bacteria in humans and animals [6]. S. agalactiae is the most important pathogenic bacteria causing mastitis in dairy cows [7]. S. agalactiae has strong infectivity isolated in the primary culture area of tilapia [8]. Human-derived S. agalactiae can cause morbidity or even death in newborns, pregnant women, the elderly, and immunocompromised people [9]. Human-derived S. agalactiae mainly causes endometritis in women and meningitis, sepsis, and pneumonia in infants and young children. Among them, the infection rate in pregnant women is higher. Previous studies have reported that about 15% to 40% of adult women carry or are infected with S. agalactiae, and 25% pass it on to babies; among them, 2% of infants show clinical symptoms, and the mortality rate can be up to 50%. Most of the surviving children have permanent neurological sequelae. The invasive neonatal infections caused by them have caused widespread concern worldwide [10–13].
Recently, with the widespread use of antibiotics, multidrug resistance has become more serious problem in S. aglactiae. High frequence of antibiotics resistance toward erythromycin, clindamycin, and tetracycline has been widely reported in S. aglactiae clinical isolates from China [14–16]. Over 30% of clinical isolates of S. agalactiae in Italy has shown the antibiotic resistance toward clindamycin and erythromycin [16–20]. Biofilm formation often enhances the bacterial resistance. Moreover, the bacteria resistance is often correlated with the virulence factors and resistance genes [20–23]. However, the relationship between resistant gene and biofilm formation of S. agalactiae is rarely reported.
Omadacycline (OMC) is a new first-in-class aminomethylcycline antibiotic against a widespectrum of gram-positive and negative aerobic bacteria, atypical and anaerobic pathogens [24–27]. OMC has been approved by FDA for the treatment of acute skin and soft tissue infections and community-acquired pneumonia [28, 29]. Susceptibility testing results in limited studies have demonstated the antimcirobial activity of OMC against a wide range of multidrug resistant gram-positive pathogens, such as methicillin-resistant S. aureus, vancomycin (Van)-resistant enterococci [8–11]. However, the the antimcirobial susceptibility of OMC against clinical isolates of S. agalactiae from China remains elusive [30]. The relationship of OMC susceptitibility with the distribution of these resistance and virulence factors in S. agalactiae remains unclear [26].
OMC belongs to a member of the tetracycline (Tet) class. Several tet-specific resistance genes, including tet (M), tet (K), tet (L), tet (A), tet (O), and tet (B), have been found to be correlated with the antibiotic resistance [25–30]. The antimicrobial activity of OMC against S. agalactiae harboring Tet-specific resistance genes in vitro has not been clarified yet [28–30].
This study aimed to evaluate the antimicrobial activity of OMC and its relationship with the resistance factors, virulence factors, and clonality of S. agalactiae from China. Here, the MIC of OMC against S. agalactiae by both microdilution were examined. The MLST, virulence factors, and resistance gene of these isolates were investigated by PCR. Biofilms were detected by crystal violet staining. The correlation between resistance and resistance genes and biofilm formation was calculated and observed.
2. Materials and Methods
2.1. Bacterial Isolates, Culture, and Chemicals
162 nonduplicate clinical S. agalactiae strains were collected from patients at Shenzhen Nanshan People's Hospital as our previous report [5]. Bacterial species were identified by standard methods using a VITEK 2 compact system (Biomérieux, Marcy l'Etoile, France). The S. agalactiae strains were cultured in 5% calf serum in TSB broth medium in a 37°C constant temperature shaker for 18–24 h and then removed. The OMC was purchased from MedChem Express (Princeton, NJ). Thetigecycline, doxycycline, minocycline, eravacylcine, radezolind, erythromycin, solithromycin, telavancin, cefprozil, tetracycline, clindamycin, chloramphenicol, and levofloxacin were purchased from Aladdin (Shanghai, China).
2.2. Antibiotic Susceptibility Testing
Antimicrobial susceptibility of OMC and several common antibiotics, including tigecycline, doxycycline, minocycline, eravacylcine, radezolind, erythromycin, solithromycin, telavancin, cefprozil, tetracycline, clindamycin, chloramphenicol, and levofloxacin, were examined by broth microdilution using the VITEK 2 compact system as before. The MICs and inhibition zone diameter of antibiotics was determined, respectively, according to the 2020 Clinical and Laboratory Standards Institute (CLSI) guidelines. The MIC breakpoints of omadacycline, tigecycline, doxycycline, minocycline, eravacylcine, radezolind, erythromycin, solithromycin, telavancin, cefprozil, tetracycline, clindamycin, chloramphenicol, and levofloxacin were selected from the 2020 CLSI. The MIC breakpoint of OMC is ≤ 0.25 mg/L as susceptible; 0.5 mg/L as intermediate; and ≥1 mg/L as resistant, which were selected following previous literature.
2.3. D-Test for Detecting the Incidence of Erythromycin Resistance to Inductively Clindamycin
We spread the S. agalactiae with a concentration of 0.5 McLaren's turbidity tube on the MH agar plate, applied erythromycin and clindamycin paper at 15 mm intervals, and placed in a constant temperature incubator at 35°C for 16–18 hours. When the bacteriostatic ring of clindamycin paper seemed to be a capital “D”, it was judged as a “D” test positive, indicating that erythromycin had induced clindamycin resistance.
2.4. qRT-PCR Analysis
qRT-PCR was used to analyze MLST and detect resistance and virulence genes. Total bacterial RNA was extracted from S. agalactiae isolates and reverse transcribed into cDNA. The Beijing Liuhe Huada Gene Company synthesized the primers for resistance and virulence genes. The cultures of the bacterial strains grew for 4 h at 37°C for overnight. Following the manufacturer instructions, we extract the genomic DNA of the strain, and store at −20°C for future use.
The system for amplification of qRT-PCR (50 μL): Dream Taq Green PCR Master Mix (2×) 25 μL, forward primers 1 μL and reverse primers 1 μL, DNA 2 μL, added ddH2O to make up to 50 μL.
qRT-PCR reaction conditions: predenaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 1 min, 30 cycles; and 72°C for 10 min.
The RNA was submitted to qRT-PCR after adding the qRT-PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA). The products of qRT-PCR were stored at 4°C. The recA served as an internal control gene. The threshold cycle (Ct) numbers were analyzed using the 2−ΔΔCt method. The qRT-PCR reaction products were electrophoresed on a 1% agarose gel and judged and analyzed according to the presence or absence of positive amplification products and the length of the target gene fragment. All qRT-PCRs were conducted in triplicate.
2.5. Crystal Violet Staining for Investigating Resistance and Virulence Gene of the Isolates Detection of Biofilms
After bacteria grew overnight in TSB medium, 200 and TSBG medium were diluted with 0.5% glucose. 200 μL per well was added to 96-well polystyrene microplates with 3 replicate wells, 37°C incubated at room temperature for 24 hours at static temperature. Next, we blotted the pores and rinsed them with PBS 3 times, fixed them with methanol for 15 minutes, stained them with 0.5% crystal violet for 10 minutes, and rinsed them with distilled water. We added 4 : 1 confluence solution of absolute ethanol and acetone, mixed evenly, and tested under OD570 photometry in the end. OG1RF and CHS787 were used as the quality control strains. The interpretation of the biofilm's OD value varies from 0.05 to 3.5 in 96 microwells after staining. Biofilm phenotype classification was based on the method of others, strong positive (OD570 > 2), moderate (OD 570, 1–2), or weak (0.5 < OD570 < 1).
2.6. Statistical Analysis
The data were analyzed using a t-test by SPSS software. P < 0.05 was considered statistically significant.
3. Results and Discussion
3.1. Antimicrobial Activity and Resistance Analysis of OMC against S. agalactiae Isolates Clinical in Vitro
The clinical S. agalactiae strains were isolated from various infective sample sources, including cervical secretions, wound secretions, ear swab, secretions, semen, venous blood, cerebrospinal fluid, pee, urethral discharge, pus, umbilical secretions, wound secretions, reproductive tract secretions, sputum, gastric juice, throat swab, eye secretions, and amniotic fluid (Figure 1). The MIC and resistance rate of those isolates against antibiotics were obtained (Table 1), and OMC showed in vitro resistance against S. agalactiae. OMC had robust antimicrobial activity against S. agalactiae in vitro, but the clinical S. agalactiae isolates exhibited a high-proportion resistance rate to minocycline, erythromycin, solithromycin, and clindamycin. The range of OMC MIC values against S. agalactiae was 0.25–1.0 mg/L. The incidence of erythromycin resistance to inductively induced clindamycin against S. agalactiae was 90.74% (Table 2), indicating that the incidence of inducing clindamycin resistance was high. The distribution of TET-specific resistance genes in clinical S. agalactiae isolates is shown in Figure 2. Our results suggest that the presence of tet (M), tet (O), tet (K), tet (M), and tet (O) genes did not affect OMC sensitivity in S. agalactiae.
Figure 1.
The clinical S. agalactiae strains were isolated from various infective sample sources from 2018 to 2019.
Table 1.
Comparison of in vitro antimicrobial activity of OMC and various common antibiotics against S. agalactiae.
| Antibiotic | No of isolates (MIC/inhibition zone diameter) | MIC (mg/L) | Resistance rate (%) | MIC (Ug/Ml) breakpoint | N | Inhibition zone diameter (mm) a breakpoint | N |
|---|---|---|---|---|---|---|---|
| Total | 162/n | — | — | — | — | — | — |
| Tigecycline | 162 | 0.06–0.5 | 0 | ≤2 | 162 | — | — |
| 4 | 0 | — | — | ||||
| ≥8 | 0 | — | — | ||||
| Doxycycline | 162 | 0.25–22 | 22.2 | ≤0.25 | 12 | — | — |
| 0.5 | 4 | — | — | ||||
| ≥1 | 110 | — | — | ||||
| Minocyclin | 162 | 0.125–32 | 82.1 | ≤4 | 23 | — | — |
| 8 | 4 | — | — | ||||
| ≥16 | 133 | — | — | ||||
| Eravacylcine | 162 | 0.015–0.25 | 0 | ≤0.25 | 162 | — | — |
| 0.5 | 0 | — | — | ||||
| ≥1 | 0 | — | — | ||||
| Omadacycline | 162 | 0.25–0.5 | 13.6 | ≤0.25 | 68 | — | — |
| 0.5 | 72 | — | — | ||||
| ≥1 | 22 | — | — | ||||
| Radezolind | 162 | 0.03–1 | 0 | ≤2 | 162 | — | — |
| 4 | -- | — | — | ||||
| ≥8 | -- | — | — | ||||
| Erytromycin | 136/25 | ≤0.06–16 | 46.3/88.0 | ≤0.25 | 66 | ≥23 | 1 |
| 0.5 | 7 | 14–22 | 2 | ||||
| ≥1 | 63 | ≤13 | 22 | ||||
| Solithromycin | 142 | 0.015–2 | 54.2 | ≤0.25 | 21 | — | — |
| 0.5 | 38 | — | — | ||||
| ≥1 | 77 | — | — | ||||
| Telavancin | 139 | 0.06–2 | — | ≤0.12 | 24 | — | — |
| — | — | — | — | ||||
| — | — | — | — | ||||
| Cefprozil | 142 | 0.004–2 | 0 | ≤2 | 43 | — | — |
| 4 | 0 | — | — | ||||
| ≥8 | 0 | — | — | ||||
| Tetracycline | 162 | — | — | ≤1 | — | — | |
| 2 | — | — | |||||
| ≥4 | — | — | |||||
| Clindamycin | 16 | — | 81.2 | — | — | ≥19 | 2 |
| — | — | 16–18 | 1 | ||||
| — | — | ≤15 | 13 | ||||
| Chloramphenicol | 39 | — | 15.4 | — | — | ≥21 | 11 |
| — | — | 18–20 | 22 | ||||
| — | — | ≤17 | 6 | ||||
| Levofloxacin | 16 | — | 25.0 | — | — | ≥17 | 8 |
| — | — | 14–16 | 4 | ||||
| — | — | ≤13 | 4 |
Notes: OMC-omadacycline, DOX-doxycycline,, MIN-minocycline, and TET-tetracycline.
Table 2.
Drug resistance rate of D-test against S. agalactiae.
| Antibiotics | N | D-test | Drug resistance rate (%) | |
|---|---|---|---|---|
| D (+) | D (−) | |||
| Omadacycline | 162 | 15 | 147 | 90.74 |
Figure 2.
The relationship among OMC, DOX, and MIN MIC distributions with ST10 (a) and ST17 (b) genotypes in S. agalactiae. Notes: OMC-omadacycline, DOX-doxycycline, MIN-minocycline, and TET-tetracycline.
3.2. Clonality of OMC MIC Distribution
Twenty-one STs were identified among the isolated S. agalactiae. The predominant STs were ST10 (32/162; 19.7%) and ST17 (20/162; 12.3%). A new type has been detected. Moreover, 18.8% of ST10 strains and 25.0% of ST17 strains had an OMC MIC level of 1 mg/L, showing clonal clustering toward the ST17 genotype (Table 3).
Table 3.
In vitro antimicrobial activity of OMC against S. agalactiae with TET-specific resistance genes.
| TET resistance gene (S) | No. | ERAV MIC (mg/L) | OMC MIC (mg/L) | RADE MIC (mg/L) | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ≤0.25 | 0.5 | ≥1 | Range | ≤0.25 | 0.5 | ≥1 | Range | ≤2 | 4 | ≥8 | Range | ||
| Tet (M) | 71 | 71 | 0 | 0 | 0.008–0.25 | 32 | 28 | 8 | 0.25–1.00 | — | — | — | — |
| Tet (O) | 46 | 46 | 0 | 0 | 0.008–0.25 | 14 | 21 | 6 | 0.25–1.00 | — | — | — | — |
| Tet (K) | 44 | 44 | 0 | 0 | 0.008–0.25 | 14 | 26 | 6 | 0.25–1.00 | — | — | — | — |
| Tet (M), tet (O) | 7 | 7 | 0 | 0 | 0.008–0.25 | 4 | 0 | 2 | 0.125–1.00 | — | — | — | — |
| Optra | 13 | — | — | — | — | — | — | — | — | 13 | 0 | 0 | 0.06–0.25 |
| None detected | 9/149 | 9 | 0 | 0 | 0.015–0.25 | 4 | 2 | 3 | 0.25–1.00 | 149 | 0 | 0 | 0.03–0.25 |
3.3. Formation Characteristics of OMC-Resistant S. Agalactiae
The 96-well plate readings after crystal violet staining ranged from 0.13 to 3.10. The median values of the OG1RF strain and the CHS787 control strain OD570 were 1.09 and 1.42, respectively. Among the S. agalactiae isolates tested, most of the S. agalactiae biofilms showed weak positive and above. Among the strains formed by these biofilms, the weak biofilm phenotypes differed from the medium biofilm phenotypes and the strong. The number of strains in the biofilm phenotype was similar.
3.4. The Correlation between Virulence and Resistance Gene and Biofilm Formation
PCR test results suggested that the OMC-resistant S. agalactiae resistance-positive genes ermB, ermC, OptrA, msrB, mefAE, tetM, tetO, and tetK were detected, but no ermA, cfr, cfrB, and msrA gene-positive strains were detected (Table 4). The relationship between drug genes and biofilm formation still needs further examination. Statistical analysis showed no significant correlation between genes and biofilm formation (P > 0.05). 136 strains of S. agalactiae resistant to OMC, analysis of PCR amplified virulence genes showed that bac, bca, fbsA, fbsB, cfb, hylB, Imb, cylE, cpsA, rib, cpsIII, PI-1, PI-2a, and PI-2b positive S. agalactiae strains were not statistically correlated with biofilm formation (P > 0.05), while the scpB gene was just the opposite of current results, and it was correlated with biofilm formation by statistical analysis (P < 0.05) (Table 5).
Table 4.
Relationships of OMC-resistant genes with biofilm formation in S. agalactiae.
| OMC-resistant genes | Number and percentage of Streptococcus agalactiae (N = 136) | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| N | Strong | Medium | Weak | Negative | All positive | P-value | ||||||
| N | % | N | % | N | % | N | % | N | % | |||
| Erma (+) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0.145744203 |
| Erma (−) | 136 | 8 | 5.88 | 14 | 10.3 | 35 | 25.7 | 79 | 58.1 | 57 | 41.9 | |
| Ermb (+) | 98 | 7 | 7.14 | 11 | 11.2 | 29 | 29.6 | 51 | 52.0 | 47 | 48.0 | 0.122867448 |
| Ermb (−) | 38 | 1 | 2.63 | 3 | 7.89 | 7 | 18.4 | 27 | 71.5 | 11 | 28.9 | |
| Ermc (+) | 2 | 0 | 0 | 0 | 0 | 0 | 0 | 2 | 1.00 | 0 | 0 | 0.146326519 |
| Ermc (−) | 134 | 8 | 5.97 | 14 | 10.4 | 35 | 26.1 | 77 | 57.5 | 57 | 42.5 | |
| Optra (+) | 13 | 2 | 15.4 | 2 | 15.4 | 2 | 15.4 | 7 | 53.8 | 6 | 46.1 | 0.863018713 |
| Optra (−) | 126 | 6 | 4.76 | 12 | 9.52 | 35 | 27.8 | 73 | 57.9 | 53 | 42.1 | |
| Cfr (+) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0.145744203 |
| Cfr (−) | 136 | 8 | 5.88 | 14 | 10.3 | 35 | 25.7 | 79 | 58.1 | 57 | 41.9 | |
| Cfrb (+) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0.145744203 |
| Cfrb (−) | 136 | 8 | 5.88 | 14 | 10.3 | 35 | 25.7 | 79 | 58.1 | 57 | 41.9 | |
| Msra (+) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0.145744203 |
| Msra (−) | 136 | 8 | 5.88 | 14 | 10.3 | 35 | 25.7 | 79 | 58.1 | 57 | 41.9 | |
| Msrb (+) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0.145744203 |
| Msrb (−) | 136 | 8 | 5.88 | 14 | 10.3 | 35 | 25.7 | 79 | 58.1 | 57 | 41.9 | |
| Mefae (+) | 30 | 6 | 20.0 | 1 | 3.33 | 3 | 10.0 | 20 | 66.7 | 10 | 33.327 | 0.094142032 |
| Mefae (−) | 106 | 29 | 27.4 | 13 | 12.3 | 32 | 30.2 | 32 | 30.2 | 74 | 69.8 | |
| Tetm (+) | 61 | 5 | 8.20 | 3 | 4.92 | 16 | 26.2 | 37 | 60.7 | 24 | 39.3 | 0.74704373 |
| Tetm (−) | 75 | 3 | 4.00 | 11 | 14.7 | 19 | 25.3 | 42 | 56.0 | 33 | 44.0 | |
| Teto (+) | 46 | 2 | 4.34 | 7 | 15.2 | 12 | 26.1 | 25 | 54.3 | 21 | 45.7 | 0.504670116 |
| Teto (−) | 90 | 6 | 6.67 | 7 | 1.11 | 23 | 25.6 | 54 | 60.0 | 36 | 40.0 | |
| Tetk (+) | 27 | 1 | 3.70 | 2 | 7.40 | 10 | 37.0 | 14 | 51.9 | 13 | 48.1 | 0.696589923 |
| Tetk (−) | 109 | 7 | 6.42 | 12 | 11.0 | 25 | 22.9 | 65 | 59.6 | 44 | 40.4 | |
Table 5.
Relationships of OMC virulence genes with biofilm formation in S. agalactiae.
| OMC virulence genes | N | Number and percentage of Streptococcus agalactiae (N = 136) | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Strong | Medium | Weak | Negative | All positive | P-value | |||||||
| N | % | N | % | N | % | N | % | N | % | |||
| Bac (+) | 136 | 8 | 5.88 | 14 | 10.3 | 35 | 25.7 | 79 | 58.1 | 57 | 41.9 | 0.145744203 |
| Bac (−) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| Bca (+) | 132 | 8 | 6.06 | 10 | 7.58 | 35 | 26.52 | 79 | 59.85 | 53 | 40.15 | 0.192189367 |
| Bca (−) | 4 | 0 | 0 | 4 | 1 | 0 | 0 | 0 | 0 | 4 | 1 | |
| Fbsa (+) | 53 | 8 | 15.1 | 12 | 22.6 | 26 | 49.1 | 7 | 13.2 | 46 | 86.8 | 0.073412483 |
| Fbsa (−) | 83 | 0 | 0 | 2 | 2.41 | 9 | 10.8 | 72 | 86.7 | 11 | 13.3 | |
| Fbsb (+) | 136 | 8 | 5.88 | 14 | 10.3 | 35 | 25.7 | 79 | 58.1 | 57 | 41.9 | 0.145744203 |
| Fbsb (−) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| Cfb(+) | 136 | 8 | 5.88 | 14 | 10.3 | 35 | 25.7 | 79 | 58.1 | 57 | 41.9 | 0.145744203 |
| Cfb (−) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| Cfr (+) | 136 | 8 | 5.88 | 14 | 10.3 | 35 | 25.7 | 79 | 58.1 | 57 | 41.9 | 0.145744203 |
| Cfr (−) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| Hylb (+) | 136 | 8 | 5.88 | 14 | 10.3 | 35 | 25.7 | 79 | 58.1 | 57 | 41.9 | 0.145744203 |
| Hylb (−) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| Imb (+) | 136 | 8 | 5.88 | 14 | 10.3 | 35 | 25.7 | 79 | 58.1 | 57 | 41.9 | 0.145744203 |
| Imb (−) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| Cyle (+) | 136 | 8 | 5.88 | 14 | 10.3 | 35 | 25.7 | 79 | 58.1 | 57 | 41.9 | 0.145744203 |
| Cyle (−) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| Cpsa (+) | 135 | 8 | 5.88 | 14 | 10.3 | 35 | 25.7 | 78 | 58.1 | 57 | 41.9 | 0.145744203 |
| Cpsa (−) | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 100 | 0 | 0 | |
| Scpb (+) | 126 | 8 | 6.35 | 14 | 11.1 | 33 | 26.2 | 71 | 56.3 | 55 | 43.7 | 0.03921902 |
| Scpb (−) | 10 | 0 | 0 | 0 | 0 | 2 | 20 | 8 | 80 | 2 | 20 | |
| Rib (+) | 96 | 7 | 7.29 | 8 | 8.33 | 23 | 24.0 | 58 | 60.4 | 38 | 39.6 | 0.552661755 |
| Rib (−) | 40 | 1 | 2.50 | 6 | 15.0 | 12 | 30.0 | 21 | 52.5 | 19 | 47.5 | |
| Cpsiii(+) | 79 | 6 | 7.60 | 4 | 5.06 | 19 | 24.1 | 50 | 63.3 | 29 | 36.7 | 0.479252866 |
| Cpsiii (−) | 57 | 2 | 3.51 | 10 | 17.5 | 16 | 28.1 | 29 | 50.9 | 28 | 49.1 | |
| Pi−1 (+) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0.145744203 |
| Pi−1 (−) | 136 | 8 | 5.88 | 14 | 10.3 | 35 | 25.7 | 79 | 58.1 | 57 | 41.9 | |
| PI−2a (+) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0.145744203 |
| PI−2a (−) | 136 | 8 | 5.88 | 14 | 10.3 | 35 | 25.7 | 79 | 58.1 | 57 | 41.9 | |
| PI−2b (+) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0.145744203 |
| PI−2b (−) | 136 | 8 | 5.88 | 14 | 10.3 | 35 | 25.7 | 79 | 58.1 | 57 | 41.9 | |
4. Conclusion
In this study, OMC exhibited robust activity resistance in vitro against S. agalactiae, and the clinical S. agalactiae isolates exhibited a high resistance rate against minocycline, erythromycin, solithromycin, and clindamycin. The main STs observed were ST10 and ST17. The data indicated the clonality of S. agalactiae with clustering of the 1 mg/L OMC MIC in the ST17 genotype. The incidence of erythromycin resistance to inductively clindamycin against S. agalactiae was higher. Therefore, the clinical microbiology laboratory must strengthen the detection of the S. agalactiae D-test, which is resistant to erythromycin and sensitive to clindamycin, to guide the rational clinical use.
The OMC resistance genes ermA, rmB [31], ermC, OptrA, msrB, mefAE, cfr, cfrB, msrA, tetM, tetO, and tetK did not show a significant correlation with biofilm formation. Analysis of PCR amplified virulence genes showed that bac, bca, fbsA, fbsB, cfb, hylB, Imb, cylE, cpsA, rib, cpsIII, PI-1, PI-2a, and PI-2b positive S. agalactiae strains have no significant correlation with biofilm formation by statistical analysis (P > 0.05) [32–35] whereas the scpB gene was just the opposite to the current result, and it was correlated with biofilm formation by statistical analysis (P < 0.05).
To sum up, OMC exhibited robust activity and in vitro resistance against S. agalactiae isolates, and OMC MIC with 1 mg/L showed ST17 clonality clustering. The incidence of erythromycin resistance to inductively clindamycin against S. agalactiae was high [36–38]. The detection of the D-test for S. agalactiae must be strengthened in clinics because of its resistance. Biofilm formation ability and OMC resistance genes ermB, ermC, OptrA, msrB, mefAE, tetM, tetO, tetK, ermA, cfr, cfrB, msrA, and virulence genes bca, bac, fbsA, fbsB, cfb, hylB, Imb, cylE, cpsA, rib, cpsIII, PI-1, PI-2a, and PI-2b showed no significant correlation. ScpB was significantly associated with biofilm formation of S. agalactiae and OMC-resistant.
Acknowledgments
This work was supported by the Natural Science Foundation of Guangdong Province (nos. 2018A0303130031 and 2020A1515011303). Thanks to the research square platform for allowing more readers to read the articles and give valuable opinions. The authors revise the articles according to the suggestions to improve the quality of the articles (https://www.researchsquare.com/article/rs-35559/v1).
Data Availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Conflicts of Interest
The authors declare that there are no conflicts of interest with this study.
Authors' Contributions
Guiqiu Li and Ying Wei contributed equally to this work.
References
- 1.Thomas L., Cook L. Two-component signal transduction systems in the human pathogen Streptococcus agalactiae. Infection and Immunity . 2020;88(7) doi: 10.1128/IAI.00931-19.e00931 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Jamrozy D., Bijlsma M. W., de Goffau M. C., et al. Increasing incidence of group B streptococcus neonatal infections in The Netherlands is associated with clonal expansion of CC17 and CC23. Scientific Reports . 2020;10(1):p. 9539. doi: 10.1038/s41598-020-66214-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Li L. M., Wu X. H., Xu L. F. Drug resistance and clinical infection distribution of Streptococcus agalactiac. Chinese Journal of Nosocomiology . 2014;24(3):549–551. [Google Scholar]
- 4.Zhang Z., Lan J., Li Y., et al. The pathogenic and antimicrobial characteristics of an emerging Streptococcus agalactiae serotype IX in Tilapia. Microbial Pathogenesis . 2018;122:39–45. doi: 10.1016/j.micpath.2018.05.053. [DOI] [PubMed] [Google Scholar]
- 5.Li P., Wei Y., Li G., et al. Comparison of antimicrobial efficacy of eravacycline and tigecycline against clinical isolates of Streptococcus agalactiae in China: In vitro activity, heteroresistance, and cross-resistance. Microbial Pathogenesis . 2020;149:e104502(1)–e104502(7). doi: 10.1016/j.micpath.2020.104502. [DOI] [PubMed] [Google Scholar]
- 6.Boonyayatra S., Wongsathein D., Tharavichitkul P. Genetic relatedness among Streptococcus agalactiae isolated from cattle, fish, and humans. Foodborne Pathogens and Disease . 2020;17(2):137–143. doi: 10.1089/fpd.2019.2687. [DOI] [PubMed] [Google Scholar]
- 7.Lavon Y., Leitner G., Kressel Y., Ezra E., Wolfenson D. Comparing effects of bovine streptococcus and Escherichia coli mastitis on impaired reproductive performance. Journal of Dairy Science . 2019;102(11):10587–10598. doi: 10.3168/jds.2019-16673. [DOI] [PubMed] [Google Scholar]
- 8.Kannika K., Pisuttharachai D., Srisapoome P., et al. Molecular serotyping, virulence gene profiling and pathogenicity of Streptococcus agalactiae isolated from tilapia farms in Thailand by multiplex PCR. Journal of Applied Microbiology . 2017;122(6):1497–1507. doi: 10.1111/jam.13447. [DOI] [PubMed] [Google Scholar]
- 9.Dalanezi F. M., Joaquim S. F., Guimarães F. F., et al. Influence of pathogens causing clinical mastitis on reproductive variables of dairy cows. Journal of Dairy Science . 2020;103(4):3648–3655. doi: 10.3168/jds.2019-16841. [DOI] [PubMed] [Google Scholar]
- 10.Chen X., Wang D., Wang T., et al. Enhanced photoresponsivity of a GaAs nanowire metal-semiconductor-metal photodetector by adjusting the fermi level. ACS Applied Materials & Interfaces . 2019;11(36):33188–33193. doi: 10.1021/acsami.9b07891. [DOI] [PubMed] [Google Scholar]
- 11.Zhang H., Du A., Hong L., Wang Y., Cheng B., Zhang Z. Identification and analysis of fluoride-resistant streptococcus mutans genomic mutation based on silicon solid nanopore. Materials Express . 2020;10(4):479–489. doi: 10.1166/mex.2020.1670. [DOI] [Google Scholar]
- 12.Su Q., Liu Y., Lv X.-W., Dai R.-X., Yang X.-H., Kong B.-H. LncRNA TUG1 mediates ischemic myocardial injury by targeting miR-132-3p/HDAC3 axis. American Journal of Physiology - Heart and Circulatory Physiology . 2020;318(2):H332–H344. doi: 10.1152/ajpheart.00444.2019. [DOI] [PubMed] [Google Scholar]
- 13.Plainvert C., Hays C., Touak G., et al. Multidrug-resistant hypervirulent group B Streptococcus in neonatal invasive infections, France, 2007-2019. Emerging Infectious Diseases . 2020;26(11):2721–2724. doi: 10.3201/eid2611.201669. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Shen D. S., Zhou X. Advances of Streptococcus agalactiae. Chinese Journal of Microecology . 2008;20(5):518–519. [Google Scholar]
- 15.Yang Y., Liu Y., Ding Y., et al. Molecular characterization of Streptococcus agalactiae isolated from bovine mastitis in Eastern China. Plos One . 2013;8(7) doi: 10.1371/journal.pone.0067755.e67755 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Nurani F. S., Sukenda S., Nuryati S. Maternal immunity of tilapia broodstock vaccinated with polyvalent vaccine and resistance of their offspring against Streptococcus agalactiae. Aquaculture Research . 2020;51(4):1513–1522. doi: 10.1111/are.14499. [DOI] [Google Scholar]
- 17.Zhu S., Wang X., Zheng Z., Zhao X., Bai Y., Liu H. Synchronous measuring of triptolide changes in rat brain and blood and its application to a comparative pharmacokinetic study in normal and Alzheimer’s disease rats. Journal of Pharmaceutical and Biomedical Analysis . 2020;185 doi: 10.1016/j.jpba.2020.113263.e113263 [DOI] [PubMed] [Google Scholar]
- 18.Liu G., Ren G., Zhao L., Cheng L., Wang C., Sun B. Antibacterial activity and mechanism of bifidocin A against Listeria monocytogenes. Food Control . 2017;73:854–861. doi: 10.1016/j.foodcont.2016.09.036. [DOI] [Google Scholar]
- 19.Su Q., Liu Y., Lv X.-W., et al. Inhibition of lncRNA TUG1 upregulates miR-142-3p to ameliorate myocardial injury during ischemia and reperfusion via targeting HMGB1- and Rac1-induced autophagy. Journal of Molecular and Cellular Cardiology . 2019;133:12–25. doi: 10.1016/j.yjmcc.2019.05.021. [DOI] [PubMed] [Google Scholar]
- 20.Zhang Z., Yang F., Li X., Luo J., Li H. Distribution of serotypes, antimicrobial resistance and virulence genes among Streptococcus agalactiae isolated from bovine in China. Acta Scientiae Veterinariae . 2019;47 doi: 10.22456/1679-9216.97254.e1699 [DOI] [Google Scholar]
- 21.Sharma D., Misba L., Khan A. U. Antibiotics versus biofilm: an emerging battleground in microbial communities. Antimicrobial Resistance and Infection Control . 2019;8(1):p. 76. doi: 10.1186/s13756-019-0533-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Lopes E., Fernandes T., Machado M. P., et al. Increasing macrolide resistance among Streptococcus agalactiae causing invasive disease in non-pregnant adults was driven by a single capsular-transformed lineage, Portugal, 2009 to 2015. Euro Surveillance: bulletin Europeen sur les maladies transmissibles . 2018;23(21) doi: 10.2807/1560-7917.ES.2018.23.21.1700473.e1700473 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Wang X., Wang J., Sun X., et al. Hierarchical coral-like NiMoS nanohybrids as highly efficient bifunctional electrocatalysts for overall urea electrolysis. Nano Research . 2018;11(2):988–996. doi: 10.1007/s12274-017-1711-3. [DOI] [Google Scholar]
- 24.Bernegossi J., Fontana C. R., Caiaffa K. S., Duque C., Chorilli M. Inhibitory effect of a KSL-W peptide-loaded poloxamer 407-based microemulsions for buccal delivery on fusobacterium nucleatum biofilm. Journal of Biomedical Nanotechnology . 2020;16(3):390–397. doi: 10.1166/jbn.2020.2896. [DOI] [PubMed] [Google Scholar]
- 25.Wang D., Chen X., Fang X., et al. Photoresponse improvement of mixed-dimensional 1D-2D GaAs photodetectors by incorporating constructive interface states. Nanoscale . 2021;13(2):1086–1092. doi: 10.1039/d0nr06788a. [DOI] [PubMed] [Google Scholar]
- 26.Di Bonaventura G., Pompilio A., Monaco M., et al. Adhesion and biofilm formation by Staphylococcus aureus clinical isolates under conditions relevant to the host: relationship with macrolide resistance and clonal lineages. Journal of Medical Microbiology . 2019;68(2):148–160. doi: 10.1099/jmm.0.000893. [DOI] [PubMed] [Google Scholar]
- 27.Bai B., Lin Z. W., Pu Z. Y., et al. In vitro activity and heteroresistance of omadacycline against clinical Staphylococcus aureus isolates from China reveal the impact of omadacycline susceptibility by branched-chain amino acid transport system II carrier protein, Na/Pi cotransporter family protein, and fibronectin-binding protein. Frontiers in Microbiology . 2019;10 doi: 10.3389/fmicb.2019.02546.e2546 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Abrahamian F. M., Sakoulas G., Tzanis E., et al. Omadacycline for acute bacterial skin and skin structure infections. Clinical Infectious Diseases: An Official Publication of the Infectious Diseases Society of America . 2019;69(1):S23–S32. doi: 10.1093/cid/ciz396. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.LaPensee K., Mistry R., Lodise T. Budget impact of omadacycline for the treatment of patients with community-acquired bacterial pneumonia in the United States from the hospital perspective. American health & drug benefits . 2019;12(1):S1–S12. [PMC free article] [PubMed] [Google Scholar]
- 30.Lin Z., Pu Z., Xu G., et al. Omadacycline efficacy against enterococcus faecalis isolated in China: in vitro activity, heteroresistance, and resistance mechanisms. Antimicrobial Agents and Chemotherapy . 2020;64(3) doi: 10.1128/AAC.02097-19.e02097 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Kondrasheva N. K., Rudko V. A., Kondrashev D. O., et al. Application of a ternary phase diagram to describe the stability of residual marine fuel. Energy & Fuels . 2019;33(5):4671–4675. doi: 10.1021/acs.energyfuels.9b00487. [DOI] [Google Scholar]
- 32.Zhu X., Lin F., Zhang Z., et al. Enhancing performance of a GaAs/AlGaAs/GaAs nanowire photodetector based on the two-dimensional electron-hole tube structure. Nano Letters . 2020;20(4):2654–2659. doi: 10.1021/acs.nanolett.0c00232. [DOI] [PubMed] [Google Scholar]
- 33.Chen Q., Ma Y., Zhao J., et al. In Vitro and in vivo evaluation of improved EGFR targeting peptide-conjugated phthalocyanine photosensitizers for tumor photodynamic therapy. Chinese Chemical Letters . 2018;29(7):1171–1178. doi: 10.1016/j.cclet.2018.04.025. [DOI] [Google Scholar]
- 34.Shi J., Luo N., Ding M., Bao X. Synthesis, in vitro antibacterial and antifungal evaluation of novel 1,3,4-oxadiazole thioether derivatives bearing the 6-fluoroquinazolinylpiperidinyl moiety. Chinese Chemical Letters . 2020;31(2):434–438. doi: 10.1016/j.cclet.2019.06.037. [DOI] [Google Scholar]
- 35.Xu Z., Zhang B., Zhong W., et al. The distribution of virulence factors in Streptococcus agalactiae and its relationship to antimicrobial resistance and MLST. Journal of Pathogen Biology . 2018;13(11):1216–1220. [Google Scholar]
- 36.Zhang Z., Cui F., Cao C., Wang Q., Zou Q. Single-cell RNA analysis reveals the potential risk of organ-specific cell types vulnerable to SARS-CoV-2 infections. Computers in Biology and Medicine . 2022;140 doi: 10.1016/j.compbiomed.2021.105092.105092 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 37.Lai W.-F., Wong W.-T. Property-tuneable microgels fabricated by using flow-focusing microfluidic geometry for bioactive agent delivery. Pharmaceutics . 2021;13(6):p. 787. doi: 10.3390/pharmaceutics13060787. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 38.Lai W.-F. Development of hydrogels with self-healing properties for delivery of bioactive agents. Molecular Pharmaceutics . 2021;18(5):1833–1841. doi: 10.1021/acs.molpharmaceut.0c00874. [DOI] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.