Omadacycline Efficacy against Streptococcus Agalactiae Isolated in China: Correlation between Resistance and Virulence Gene and Biofilm Formation

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.


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.
ere 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. e invasive neonatal infections caused by them have caused widespread concern worldwide [10][11][12][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][15][16]. Over 30% of clinical isolates of S. agalactiae in Italy has shown the antibiotic resistance toward clindamycin and erythromycin [16][17][18][19][20]. Biofilm formation often enhances the bacterial resistance. Moreover, the bacteria resistance is often correlated with the virulence factors and resistance genes [20][21][22][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 grampositive and negative aerobic bacteria, atypical and anaerobic pathogens [24][25][26][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][9][10][11]. However, the the antimcirobial susceptibility of OMC against clinical isolates of S. agalactiae from China remains elusive [30]. e relationship of OMC susceptitibility with the distribution of these resistance and virulence factors in S. agalactiae remains unclear [26].
is 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. e MLST, virulence factors, and resistance gene of these isolates were investigated by PCR. Biofilms were detected by crystal violet staining. e correlation between resistance and resistance genes and biofilm formation was calculated and observed.
e MICs and inhibition zone diameter of antibiotics was determined, respectively, according to the 2020 Clinical and Laboratory Standards Institute (CLSI) guidelines. e MIC breakpoints of omadacycline, tigecycline, doxycycline, minocycline, eravacylcine, radezolind, erythromycin, solithromycin, telavancin, cefprozil, tetracycline, clindamycin, chloramphenicol, and levofloxacin were selected from the 2020 CLSI. e 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.

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.

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. e Beijing Liuhe Huada Gene Company synthesized the primers for resistance and virulence genes. e 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.
e RNA was submitted to qRT-PCR after adding the qRT-PCR Master Mix ( ermo Fisher Scientific, Waltham, MA, USA). e products of qRT-PCR were stored at 4°C. e recA served as an internal control gene. e threshold cycle (Ct) numbers were analyzed using the 2−ΔΔCt method. e 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.

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. e 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).

Statistical Analysis.
e data were analyzed using a t-test by SPSS software. P < 0.05 was considered statistically significant.

Antimicrobial Activity and Resistance Analysis of OMC against S. agalactiae Isolates Clinical in Vitro.
e 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). e 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 highproportion resistance rate to minocycline, erythromycin, solithromycin, and clindamycin. e range of OMC MIC values against S. agalactiae was 0.25-1.0 mg/L. e 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. e distribution of TET-specific resistance genes in clinical S. agalactiae isolates is shown in Figure 2 (Table 3).

Formation Characteristics of OMC-Resistant S. Agalactiae.
e 96-well plate readings after crystal violet staining ranged from 0.13 to 3.10.
e 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. e number of strains in the biofilm phenotype was similar.

e 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 genepositive strains were detected (Table 4). e 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  Computational Intelligence and Neuroscience 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).

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. e main STs observed were ST10 and ST17. e data indicated the clonality of S. agalactiae with clustering of the 1 mg/L OMC MIC in the ST17 genotype. e incidence of erythromycin resistance to inductively clindamycin against S. agalactiae was higher. erefore, 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. e 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][33][34][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).
Data Availability e datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Conflicts of Interest
e authors declare that there are no conflicts of interest with this study.