Experimental Study on Streptococcus agalactiae Genotype and Erythromycin Resistance in Neonatal Sepsis

This study aimed to evaluate Streptococcus agalactiae genotype and erythromycin resistance in neonatal sepsis. After obtaining the mothers’ informed consent, trained nurses sampled 430 neonatal specimens of sepsis from the ear canal, oral cavity and umbilical cord immediately after childbirth and implemented a cross-sectional study. By Gram staining, morphology, hemolysis mode, catalase and CAMP tests, the isolate was identified as S. agalactiae. All 455 isolates were tested for antimicrobial susceptibility by the disc diffusion method. Multilocus sequence typing was used to serotype S. agalactiae involving sequencing of 7 housekeeping genes. The erythromycin resistance genes-erm (B), erm (A) and mef (A) were detected by PCR. Results showed that there were 286 cases (66.51%) of neonates delivered naturally, and 144 cases (33.49%) of neonates delivered by cesarean section. A total of 455 strains were tested, including 253 strains (55.60%) of Gram-positive bacteria with 100 strains (21.98%) of S. agalactiae and 52 strains (11.43%) of Staphylococcus epidermidis, 178 strains (39.12%) of Gram-negative bacteria with 45 strains of Klebsiella pneumoniae (9.89%), 36 strains of Escherichia coli (7.91%), 36 strains of Pseudomonas aeruginosa (7.91%), and 323 strains of Citrobacter freundii (7.03%). S. agalactiae had the highest resistance of 87 (87.00%) to erythromycin, followed by resistance to azithromycin 83 (83.00%) and clindamycin 78 (78.00%). In children with neonatal sepsis, S. agalactiae serotypes were mainly Ia, Ib, and III, accounting for 29.00%, 35.00%, and 19.00% respectively. The main genotypes were ST651, ST103 and ST176, which account for 19.00%, 17.00% and 15.00% respectively. The ST19 type 13.00%, ST27 type 8.00%, ST17 Type 11.00%, ST10 type 12.00%, ST485 type 5.00%. The ST103 and ST485 isolates were classified as serotype Ia, the ST10 and ST176 isolates were classified as serotype Ib, and ST17 and ST19 isolates were classified as serotype III. Among the strains of S. agalactiae, 40.23% (35/87) carry erm (A) gene, 35.63% (31/87) carry erm (B) gene, and 24.14% (21/87) carry mef (A) gene. erm (A) gene was the most common gene in ST19 strain (7/11, 63.64%), and erm (B) gene was the most common gene in ST176 and ST651 strains (6/12, 50.00%; 8/18, 44.44%), while mef (A) gene was the most common gene in ST17 strain (5/11, 45.45%). In general, S. agalactiae genotypes in neonatal sepsis were mainly ST651, ST103 and ST176, and the main serotypes are Ia, Ib, and III. There was good consistency between ST and serotype, and a significant difference was shown in erythromycin resistance and ST distribution, which highlights the value of new epidemiological trend detection by monitoring multiple characteristics and provides inspiration for the development of multivalent S. agalactiae vaccines.


Introduction
Streptococcus agalactiae as an important human pathogen that causes neonatal pneumonia, sepsis and meningitis may appear in the form of early or lateonset in neonates and infants (1). S. agalactiae is still the main cause of neonatal mortality and morbidity in many parts of the world (2). S. agalactiae occupies gastrointestinal and reproductive tracts of approximately 25% of healthy pregnant women, which will be passed to neonates during delivery (3). Colonization of S. agalactiae has a relation with increased risk of nosocomial infections and community-related infections of the organism (4). The capsular polysaccharide of S. agalactiae has chemical and antigenic differences, which can be subdivided into ten serotypes, namely Ia, Ib, and II-IX (5). Vaccines against S. agalactiae infection must include more common serotypes associated with the disease in different populations (6). The epidemiological distribution of these serotypes may vary in several aspects, including geographic area, the profile of the study population, and the source of bacterial isolates (7,8). In the United States and certain European countries, the four serotypes (Ia, II, III and V) are usually the most common isolates (9). Several studies conducted in Brazil have described the production of serotypes Ia, Ib, II, III, IV and V (10). Penicillin or amoxicillin is used for chemoprevention during childbirth and has been used in pregnant women with risk factors for vaginal S. agalactiae in order to reduce the incidence of early-onset diseases in neonates (11). Erythromycin is recommended for prenatal prophylaxis of S. agalactiae in women who are at high risk of penicillin allergy or treatment failure (12). The main purpose of this study is to evaluate the S. agalactiae genotype and erythromycin resistance in neonatal sepsis.

Materials and methods Study population and sample collection
This cross-sectional study was conducted on August 15th, 2018. The project was approved by the research ethics committee of the hospital. After obtaining the mothers' informed consent, trained nurses sampled neonatal specimens of sepsis from the ear canal, oral cavity and umbilical cord immediately after childbirth. The culture was carried out using conventional methods, and the isolate was identified as S. agalactiae.

Identification of S. agalactiae
During transportation, the swab was inoculated into a selective medium (Trans-Vag broth containing 8μg/mL gentamicin and 15μg/mL nalidixic acid) at 4°C, and further inoculated for 24 hours at 37°C under 5% CO 2 . Then subculture a circle of broth onto 5% sheep blood agar and incubate at 37°C, 5% CO 2 for 24 hours. Through a combination of Gram staining, morphology, hemolysis mode, catalase test and Christie Atkins Munch Peterson (CAMP) test, the isolate was identified as S. agalactiae.

Antimicrobial susceptibility test
According to the CLSI guidelines, all 455 isolates were tested for sensitivity to erythromycin, azithromycin, clindamycin, amoxicillin, cefazolin, penicillin, levofloxacin, tetracycline and teicoplanin by disc diffusion method. The resistance phenotype was determined by the erythromycin-fosfomycin double-disc test.

Serotype analysis
Multi-locus sequence typing was used for serotyping of S. agalactiae involving sequencing of 7 housekeeping genes. Sequence types (STs) and alleles were confirmed by database inquiry (http://eburst.mlst.net). By using the eBURST software program (Department of Epidemiology of Infectious Diseases, Imperial College London, UK; London, UK; http://eburst.mlst.net), cluster ST into clonal complexes (CC). Serotype all the isolates by HCl extraction method and Double immunodiffusion test or capillary precipitation test. The capsular polysaccharides (Ia, Ib and II-V) prepared indoors by standard methods were subjected to antiserum typing. The isolates that cannot be typed are referred to as NT.

Determination of erythromycin resistance gene
Screen all strains for erythromycin resistance genes. erm (B), erm (A) and mef (A) genes were detected by PCR amplification using the aforementioned primers and subjected to minor modifications, and properties of PCR products were checked by sequencing. In short, PCR has a final volume of 25 mL, which contains PCR buffer, 0.2 mM of each deoxynucleoside triphosphate, 0.5 mM of each primer, 2 IU of Taq polymerase and 25 ng of template DNA. The cycling conditions are as follows: initial denaturation at 948°C for 2 minutes, then cycling at 94°C for 1 minute, 55°C for 1 minute and 72°C for 1 minute for 35 cycles (finally extended at 72°C for 10 minutes). The products were separated by electrophoresis in 1.6% agarose gel in TBE (8.9 mM Tris, 8.9 mM boric acid, 0.25 mM EDTA; pH 8.0) buffer. S. pyogenes CIP107003, S. pyogenes CIP107002 and S. agalactiae 03-226 were used as positive controls for erm (B), erm (A) and mef (A) genes, respectively.

Data analysis
All data were entered into EpiData 3.1 database in duplicate. The minimum spanning tree illustrates the relationship between STS and the serotype of S. agalactiae isolates. Categorical variables were compared by Fisher's exact test. These analyses were performed using STATA version 13.0. P<0.05 indicates a statistically significant difference.

Results and discussion General information statistics
This study involved 430 children with neonatal sepsis. The male to female ratio was 228:202, the average gestational age was 38.26±1.57, and the mother's gestational age was 27.16±3.19. There were 286 cases (66.51%) of neonates delivered naturally, and 144 cases (33.49%) of neonates delivered by cesarean section. All the children had symptoms within 3 weeks of birth. There were 126 cases of neonatal hyperbilirubinemia, 157 cases of fever, 134 cases of shortness of breath, and 13 cases of congenital heart disease. (Table 1)

Analysis of strain drug resistance to antibiotics
A total of 455 strains were detected in children with neonatal sepsis. The strains were cultured and tested for resistance. Where, S. agalactiae has the highest resistance of 87 (87.00%) to erythromycin, followed by resistance to azithromycin 83 (83.00%) and clindamycin 78 (78.00%). Other strains have the highest drug resistance of 167 (47.04%) to tetracycline, followed by resistance to clindamycin 128 (36.06%). (Table 3)  24 5.27%

Relationship between serotype and genotype of S. agalactiae in neonatal sepsis
The relationship between serotype and genotype of S. agalactiae in children with neonatal sepsis was studied. The results show good consistency between certain ST and serotypes. The ST103 and ST485 isolates are classified as serotype Ia, the ST10 and ST176 isolates are classified as serotype Ib, and ST17 and ST19 isolates are classified as serotype III. (Figure 2, Table 6)   Genotype  n  Ia  Ib  II  III  IV V/NT  ST19  13  0  0  0  13  0  0  ST27  8  0  0  8  0  0  0  ST176  15  0  15  0  0  0  0  ST17  11  0  0  0  6  3  2  ST651  19  7  8  0  0  2  2  ST103  17    S. agalactiae is an important cause of neonatal sepsis and meningitis. In the past decade, S. agalactiae has an increasingly higher correlation with invasive disease in non-pregnant adults (13).
Serotype determination has traditionally been used in epidemiological studies of S. agalactiae, which greatly helps the development of a broadly protected vaccine that contains capsular polysaccharides or polysaccharides bound to proteins (14,15). The distribution of serotypes varies by region. Studies conducted in European countries, the United States and Latin America have shown that serotypes Ia, Ib, III or V are usually the most common (16). Type III and type V serum predominate in infection cases of neonates and non-pregnant adults, while in Portugal, type Ia serum predominates in S. agalactiae-induced invasive infections in non-pregnant adults (17). In Brazil, previous studies have shown that serotypes Ia, II, III and V are found in 68.2% isolates, mainly from vaginal specimens of asymptomatic pregnant women (18). In this study, serotypes Ia, Ib, and III were the most common, and serotypes VI to VIII were not found.
In our study, the prevalence of S. agalactiae colonies in infants was 1.3%, similar to that of Japan (1.3%), but lowers than that in Bangladesh (6.3%), Turkey (17.3%) and Gambia (24.8%) of previous research. Colonization of S. agalactiae exhibits significant differences between different populations, which may be related to race, ethnicity, and genetic composition (19). It is worth noting that the prevalence is high in Italy (21.9%, sampling from ear, throat and rectum) and Gambia (24.8%, sampling from nasopharynx and rectum), but low in Pakistan (3.1%, sampling from abdominal skin and ears) and Greece (2.4%, sampling from the ear canal, throat and umbilical cord) (20). These findings indicate the importance of sampling points in determining the colonization of neonatal S. agalactiae. Only sampling from the skin and mucous membrane surfaces (such as the ear canal, oral cavity and umbilical cord) may underestimate the true prevalence of S. agalactiae in our study. It is worth noting that neonatal specimens of S. agalactiae were taken immediately after birth, indicating that the sampling was performed via vertical transmission of the mother.
Penicillin is a first-line drug recommended for the prevention and treatment of S. agalactiae disease.
Similar to the latest meta-analysis, 97% of all S. agalactiae isolates in this study are susceptible to penicillin infection, indicating that penicillin is still the first choice for prevention. It is recommended to use erythromycin and clindamycin as alternatives, especially in neonates allergic to penicillin. The resistance rates to erythromycin (87.00%) and clindamycin (78.00%) were similar to previous domestic reports (74.1%-92.5% and 64.2%-87.5%, respectively). In addition, the latest meta-analysis reveals that the resistance rate to clindamycin and erythromycin is significantly higher in Asia than in non-Asian countries. These findings suggest that alternative treatments for neonates intolerant to penicillin should be guided by the antibiotic resistance model. We observed that approximately 66.00% of isolates were multi-drug resistant, and the main multidrug resistance model featured resistance to erythromycin, azithromycin and clindamycin. Consistent with our research, reports of invasive S. agalactiae isolates from neonates in China indicate that 90% are multi-drug resistant, and these isolates show drug resistance to erythromycin, clindamycin and tetracycline. According to research on S. agalactiae-induced community and hospital-acquired infections in non-pregnant women, Chinese adults have an overall drug resistance rate of 23%, but the drug resistance model remains unclear. These findings highlight the importance of continuous monitoring of antibiotic resistance.
As far as we know, for S. agalactiae isolates, there is a rare mechanism of drug resistance. Regarding the macrolide resistance genes, erm gene modifies the ribosome through methylation and inhibits erythromycin binding, while mef gene encodes a membrane protein that acts as a drug pump (21). In our study, the most common erythromycin resistance genes are erm (A), erm (B) and mef (A). Similarly, the high prevalence of erm (A) and erm (B) has also been reported in South Korea and Barcelona (22). erm (A) gene is the most common gene in ST19 strain (7/11, 63.64%), and erm (B) gene is the most common gene in ST176 and ST651 strains (6/12, 50.00%; 8/18, 44.44%), while mef (A) gene is the most common gene in ST17 strain (5/11, 45.45%). A Spanish study found that the correlation between these genes in S. pneumoniae may be caused by the presence of a conjugative transposon that encodes erm(B) and tetM, but the underlying relationship and mechanism in S. agalactiae remain unclear (23). Future research must pay more attention to the genetic mechanism of resistance of S. agalactiae isolates worldwide to antibiotics.
Our study found that the most common ST is ST651, followed by ST103 and ST176, which is similar to the results of France, Italy and Poland (24,25). However, there are various reports about other antibiotic-resistant bacteria such as Escherichia coli, Staphylococcus aureus, and Acinetobacter baumannii strains (26,27), but there are little data on the differentiation of S. agalactiae clones based on phenotype and molecular characteristics (28)(29)(30). We observed good consistency between certain ST and serotypes, such as ST17/ST19 and III serotypes, ST103/ST485 and Ia serotype, as well as ST10/ST176 and Ib serotype. The latest research on invasive infection of S. agalactiae also reveals good consistency between certain ST and serotypes (for example, ST19 and III, ST23 and Ia, ST12 and Ib, ST1 and V).
In summary, we distinguished S. agalactiae clones in neonatal sepsis by using a variety of phenotypes and molecular characteristics and found a specific phenotype-genotype combination of the S. agalactiae clones. S. agalactiae genotypes in neonatal sepsis are mainly ST651, ST103 and ST176, and the serotypes are mainly Ia, Ib, III. There is good consistency between ST and serotype, and a significant difference is shown in erythromycin resistance and ST distribution, which highlights the value of new epidemiological trend detection by monitoring multiple characteristics and provides inspiration for the development of multivalent S. agalactiae vaccines. In addition, our findings indicate a high prevalence of multi-drug resistant S. agalactiae, which highlights the importance of continuous monitoring of antibiotic resistance.