Emergence of Invasive Serotype Ib Sequence Type 10 Group B Streptococcus Disease in Chinese Infants Is Driven by a Tetracycline-Sensitive Clone

Background Group B streptococcus (GBS) is a leading cause of serious infections in infants. The extensive use of tetracycline has led to the selection of specific resistant and infectious GBS clones. The sequence type (ST) 10 GBS strain, causing invasive infections in infants, is becoming prevalent in China. We aimed to understand the clinical and microbiological characteristics of this GBS strain. Methods We conducted a retrospective study on infants with invasive GBS disease from the largest women’s and children’s medical center in Shanxi and collected data between January 2017 and October 2020. GBS isolates were analyzed by capsule serotyping, genotyping, antibiotic resistance, and surface protein genes. Results All ST10 isolates belonged to serotype Ib; type Ib/ST10 strains were responsible for 66.7% (14/21, P < 0.05) of infant invasive GBS infections during the period and all resulted in late-onset (LOD) and late LOD disease (14/14). Infants with type Ib/ST10 GBS disease had significantly higher rates of meningitis (9/14, 64.3%, p < 0.05) and clinical complications (5/14, 35.7%, p < 0.05). The Ib/ST10 GBS isolates had limited genetic diversity, clustered in the CC10/bca/PI-1 + PI-2a genetic lineage, showed resistance to erythromycin, lincomycin, and fluoroquinolones and sensitivity to tetracycline, and possessed genes ermT, ermB, and amino acid changes in gyrA and parC. Conclusions The probable clonal expansion can result in severe infections in infants and ongoing emergence of multi-drug resistant isolates. Continued monitoring for type Ib/ST10 GBS infections is warranted.


INTRODUCTION
Group B streptococcus (GBS) is a leading cause of serious infection in young infants, commonly resulting in sepsis, pneumonia, focal infections, bacteremia, and meningitis, which leads to a serious death rate Madrid et al., 2017). GBS infections can be further divided based on onset: early onset (EOD; birth to 7 days), late-onset (LOD; 8 to 90 days), and late LOD disease (LLOD; older than 3 months of age). The current gold standard for preventing the transfer of GBS from mother to neonate in many countries is the use of intrapartum antibiotic prophylaxis during delivery Li et al., 2017;Furfaro et al., 2018). Although this precaution has reduced the incidence of GBS-associated EOD, the rates of LOD with GBS infections remain unchanged.
Conventionally, descriptive GBS epidemiology is based on capsular serotyping or genotyping Madrid et al., 2017;Furfaro et al., 2018). Ten serotypes (Ia, Ib, and II-IX) have been identified according to their capsular polysaccharide antigens, and five of them (Ia, Ib, II, III, and V) occupied approximately 96% of all neonatal invasive GBS infections globally. Multilocus sequence typing (MLST) has been used to target seven conserved housekeeping genes in classified GBS strains into numerous sequence types (STs) and clonal complexes (CCs), of which ST1, ST10, ST17, ST19, and ST23 are more frequently associated with invasive infection. Furthermore, clustered regularly interspaced short palindromic repeats (CRISPRs) have been recently identified in most archaea and many bacteria (Gholizadeh et al., 2017;Koonin and Makarova, 2019;Westra et al., 2019). This technique is a novel and effective way to reliably research population-wide GBS genomics (Sorek et al., 2008;Hidalgo-Cantabrana et al., 2018).
The general role of pathogens as the world's leading cause of neonatal disease is a concern for antibiotic resistance in GBS strains and the selective pressures we have imposed through extensive use of antibiotic drugs, such as tetracycline (Da Cunha et al., 2014;Lawn et al., 2017;Madrid et al., 2017). Penicillin is a medication of choice for prophylaxis and GBS disease therapy, with erythromycin and clindamycin recommended for patients with a ß-lactam allergy. However, second-line resistance to antibiotics continues to be strong, and the production of new phenotypes in GBS populations is observed. Even worse, reports of multi-drug resistant isolates are becoming more prevalent (Campisi et al., 2016;Guo D. et al., 2018;Hayes et al., 2020).
III/ST17 GBS strains have been particularly well studied given their role as the major cause of severe, invasive infant disease (Tazi et al., 2010;Furfaro et al., 2018;Ratner, 2019;Tazi et al., 2019). We recently reported a high incidence of isolation for both carriage and invasive ST10 GBS in Shanxi (Zhang et al., 2020), Northern China. Ji et al. also described that the percentage of invasive GBS cases caused by serotype Ib (93.4% were ST10) was higher in both the Northern and Northeastern regions of China compared with others (Ji et al., 2019). Despite the clear and growing impact of ST10 GBS strains, there remains limited data concerning the clinical and microbiological properties of this GBS strain.
Here, we aimed to (i) describe the recent ST10 manifestations and outcomes of infants with invasive GBS disease, and (ii) investigate the microbiological characteristics of the ST10 GBS strain by determining its capsule serotyping, CRISPR profiles, antibiotic resistance, and surface protein genes.

MATERIALS AND METHODS
The study protocol was authorized by the ethics committee of Shanxi Children's Hospital Shanxi Maternal and Child Health Hospital. Neither study-specific nor any other measures are needed for this study, and the data were analyzed in anonymity. Accordingly, the ethics committee waived the provision for written informed consent by registered patients.

Study Design and Strain Collection
We isolated GBS strains (n = 21) from infants with invasive GBS disease from January 2017 to October 2020. These cases were found retrospectively from the database of Shanxi Children's Hospital Shanxi Maternal and Child Health Hospital, a tertiary teaching hospital that houses 1,600 beds.
We classified invasive GBS disease sepsis, meningitis, pneumonia, or focal infections based on tests and diagnoses of laboratories reported in the medical registers of the patients. Sepsis was characterized as the bacterial increase of GBS in the blood drawn from the peripheral vein and with symptoms associated with sepsis. Meningitis was defined as the presence of clinical symptoms with positive results on cerebrospinal fluid (CSF) culture or sepsis with CSF pleocytosis in patients with a negative CSF culture. Pneumonia was defined as the presence of clinical symptoms with radiological findings and positive results on sputum or bronchoalveolar lavage fluid culture. Focal infection disease was defined as the presence of clinical symptoms and positive results for pyogenic fluids and tissue culture. Diseases between 0 and 7 days and 8 to 90 days respectively were described as EOD and LOD. The disease was listed as LLOD after 90 days of age Madrid et al., 2017).
The corresponding medical professionals undertook a retrospective chart analysis. We collected the following: demographic data and information on the clinical and laboratory observations, presence of meningitis, antimicrobial susceptibility of the pathogen, and clinical outcomes at discharge.

Capsule Serotyping and MLST
A multiplex PCR assay for identifying serotypes Ia to IX of GBS developed by Imperi et al. (2010) was conducted. The PCR is amplified and sequenced to seven housekeeping genes (adhP, pheS, atr, glnA, sdhA, glcK, and tkt) (Jones et al., 2003). The allele, ST and CC research was conducted in the MLST database (https://pubmlst.org/organisms/streptococcus-agalactiae) and a goeBURST program. tetracycline, and linezolid was measured by performing Kirby-Bauer disk diffusion according to the Clinical and Laboratory Standards Institute (Clinical and Laboratory Standards Institute, 2020).

CRISPR Analysis
The CRISPR1 loci were amplified and sequenced as previously described Beauruelle et al., 2017). CRISPR1 regions were amplified by using the oligonucleotide pairs CRISPR1-PCRF/ CRISPR1-PCRR. Then PCR products were sequenced by using internal sequencing primers CRISPR-SEQF/CRISPR-SEQR. CRISPR sequences were stored and analyzed by using the CRISPRs web server (https://crispr.i2bc.paris-saclay.fr/). This site acts as a gateway to publicly accessible CRISPRs database and software. It enables the easy detection of CRISPRs in locally produced data and consultation of CRISPRs present in the database. As a result, the CRISPR1 loci were divided into direct repeats and corresponding spacer sequences. Each unique spacer was numbered manually, and the result was analyzed and modified according to Lier et al. (2015).

Phylogenetic and Statistical Analysis
A neighbor-joining phylogenetic tree with an interior-branch test was constructed using MEGA-X. Analyses were conducted via SPSS software, v. 19.0. A Student's t-test, chi-square test, and Fisher's exact test were used for comparing proportions when necessary. All statistical testing was two-sided, and P <0.05 was set in significance.

Isolates
Over three years, a total of 21 invasive GBS diseases have been reported, including 16 isolates recovered from blood and five from cerebrospinal fluid (Figure 1).

Clinical Manifestations in Type Ib/ST10 GBS Strain Infection
These invasive GBS diseases are summarized in clinical presentations in Table 3.

Surface Protein Gene Profile and Pili
In genes encoding a-C protein and a-like proteins (Alps), we found discrepancies between isolates ( Figure 1): ST1 isolates possessed gene alp3, encoding Alp3. The ST10 strains had gene bca, encoding a-C protein, the ST19, and ST17 isolates possessed the gene encoding Rib protein, and the ST23 strains had the gene encoding the epsilon protein. The PI of GBS had three types: PI-2a, PI-2b, and PI-1 + PI-2a. All ST17 were PI-2b, all ST23 were PI-2a. All ST10, ST19, and ST1 were PI-1 + PI-2a. Figure 2 indicates that there is a link between serotypes and resistance-virulence. All isolates were susceptible to penicillin G,

Antimicrobial Resistance Genes Present in GBS Strains
All lincosamide-and macrolide-resistant strains, including all Ib/ ST10 strains (Figure 2), possessed gene ermB and ST19 isolates also include genes mefA and lnuB. The tetM and tetO genes have been linked with tetracycline resistance, and they were carried by all invasive GBS isolates except for type Ib/ST10 strains. All FQresistant isolates (Ib/ST10, III/ST19, and V/ST1) had amino acid changes within the quinolone resistance-determining region (QRDR) of gyrA and parC compared with the reference sequence. The mutations were S79F (15/17) or S79Y (2/17) in the product of parC and S81L in gyrA (17/17).

CRISPR Analysis and Phylogenetic Reconstruction
A CRISPR1 research was carried out to further study the genetic ties between invasive GBS isolates. As shown in Figure 3, except for three strains, 377 failed to revive in later testing of CRISPR1; 492, and 678 had sequence gaps at CRISPR1. All the other 18 strains had intact CRISPR1 sequence structures. The CRISPR sequences of ST10 strains mostly contained No. 02-022 spacers, compared with that of other strains.

DISCUSSION
Numerous research studies on GBS molecular epidemiology and genomic diversity have been carried out over the past few  decades Li et al., 2017;Madrid et al., 2017;Furfaro et al., 2018). However, data relating to the genetic features of the prevalent circulating lineages and in particular geographical areas remain scarce. Here, we characterized a collection of 14 invasive type Ib/ST10 GBS strains isolated from infants with invasive GBS disease in Shanxi, China during 2017-2020. Serotype Ib GBS infections are more prevalent in Asia than in other regions of the world Li et al., 2017;Madrid et al., 2017;Furfaro et al., 2018). In Japan, 19.8% of pregnant women and 34.9% of asymptomatic neonates from 2006 to 2009 were confirmed to be Ib GBS serotypes (Wakimoto et al., 2011). In South Korea, the GBS serotype Ib accounted for 22% of infections in adults (Uh et al., 2005). In Taiwan, Lin et al. found that serotype Ib GBS accounted for 21.6% of the isolates collected from a mixed population of pregnant women, neonates (Lin et al., 2014), and non-pregnant adults, indicating the commonality of the serotype. Similarly, in China, Ji et al. (2019) reported that the serotype Ib strains were the second most common cause of invasive infant disease (28.7%) from 2015 to 2017, particularly in the Northern and Northeastern regions. The 66.7% serotype Ib GBS prevalence in this study indicates that the diffusion of serotype Ib in China is a significant pathogen. To our knowledge, this is the highest level of infant invasive Ib GBS serotype disease ever recorded.
Previously, we have recorded that a high incidence of ST10 strains among both infants and colonized pregnant women (Zhang et al., 2020) strongly correlated with serotype Ib. The GBS MLST method was developed by Jones and colleagues using seven housekeeping genes (adhP, atr, glcK, glnA, pheS, sdhA, and tkt) (Jones et al., 2003). Associations between sequence type (ST) and serotype have been previously reported Li et al., 2017;Madrid et al., 2017;Furfaro et al., 2018), with varying levels of correlation. Observed trends reveal variation between serotype Ib and II within ST10 (Kang et al., 2017;Lo et al., 2019), with serotype Ib mostly associated with CC10, including ST8, ST10, and ST12. While it has been suggested that there could be a horizontal transfer of capsular genes in the GBS population (Luan et al., 2005), we observed that all serotype Ib strains belonged to CC10 ST10.
CRISPRs, the bacterial adaptive immune defense mechanism used to defend against foreign nucleic acids, are a family of noncontiguous DNA repeats interspersed by unique spacer sequences of constant length (Gholizadeh et al., 2017;Koonin and Makarova, 2019;Westra et al., 2019). The phylogenetic relations between the subspecies can be studied using CRISPR analyzes (Sorek et al., 2008;Hidalgo-Cantabrana et al., 2018). The presence of two GBS CRISPR systems: CRISPR1 and CRIPR2 has been recorded, in which CRISPR1 was active due to its high diversity of repeat arrays (Lopez-Sanchez et al., 2012). Furthermore, CRISPR1 sequences in GBS represent a new and effective method to accurate population genomics research for this species and to gain a better understanding of the dissemination of specific clones. In our study, we found that the CRISPR1 sequences of type Ib/ST10 GBS strains were highly conserved, indicating the limited genetic diversity of the strains.
We also reported the clinical manifestations of type Ib/ST10 invasive GBS strains in infants. For the majority of LOD and LLOD cases in our community, Type Ib/ST10 GBS was the most dominant invasive clone. The adaptation of unique GBS clonal complexes to a particular niche(s) was not without precedent. The CC17 lineage of GBS (mostly composed of clonal serotype III strains) is widely recognized to have been adapted to the neonatal niche, particularly late-onset neonatal disease (Musser et al., 1989). Additionally, more than 90% (210/229) of the invasive serotype V isolates were also closely associated with ST1 clone isolates between 1992 and 2013 (Flores et al., 2015). Furthermore, another expansion of a single clone causing human disease, ST283, has been identified in Southeast Asia (Barkham et al., 2019). Here, we notice that type Ib/ST10 clonal strains are also expanding rapidly among infant niches, particularly in the late onset neonatal invasive infection niche. Serotype Ib GBS strains are associated with more severe clinical presentations (Ji et al., 2019;Kao et al., 2019), including higher rates of septic shock, disseminated intravascular coagulopathy, and multiorgan failure, leading to higher mortality. We found that out  Penicillin is the medicine preferred for the prevention and treatment of GBS disease, with macrolides such as erythromycin and lincosamides, such as clindamycin, recommended for patients with a ß-lactam allergy (Li et al., 2017;Furfaro et al., 2018;Hayes et al., 2020). Although several studies revealed high macrolide resistance rates across serotype Ib GBS strains globally (Guo D. et al., 2018;Hayes et al., 2020), we found that the erythromycin resistance rate of type Ib/ST10 (100%) strains was higher than reported. In GBS, FQ resistance emerged in the early 2000s and continues to grow around the world, with rates reaching up to 40% in Asian countries (Wehbeh et al., 2005;Wang et al., 2013;Hays et al., 2016;Wu et al., 2017;Hayes et al., 2020). FQ resistance is predominantly associated with CC-10 and CC-19 isolates, which are conferred by point mutations in the QRDR, mostly at codon position 81 in gyrA and 79 or 83 in parC. We observed that 100% type Ib/ST10 strains were resistant isolates and had amino acid changes within the QRDR of gyrA and parC compared with the reference sequence. Thus, all Ib/ ST10 GBS isolates in our study had multi-drug co-resistance to erythromycin, clindamycin, and levofloxacin. FQ resistance was of limited clinical relevance here, given that FQ is contraindicated in neonates and their expectant mothers who form the largest patient groups. However, one cannot exclude the possibility of selection, fixation, and worldwide dissemination of highly epidemic multi-drug-resistant GBS clones through the extensive use of FQ, as the extensive use of tetracycline has specifically selected GBS clones infecting humans in the 1970s (Da Cunha et al., 2014;Hays et al., 2016).
Most GBS strains isolated from humans are reported to be resistant to tetracycline, and in particular, the acquisition of resistant elements, tetO, and tetM, by a subset of GBS clones, has led to their selection and expansion (Da Cunha et al., 2014;Hayes et al., 2020). Expansion of this GBS clone has led to the emergence of strains more adapted to colonizing and infecting humans. Particularly, this has led to the dissemination of the CC17 hypervirulent clone, which is associated with invasive neonatal infections. However, in our study, it is noteworthy that all type Ib/ST10 strains analyzed were sensitive to tetracycline. To investigate the tetracycline resistance rate of type Ib/ST10 GBS strains in public literature, we detailed the documented cases worldwide (Table 4) (Kang et al., 2017;Martins et al., 2017;Wu et al., 2017;Simoni et al., 2018;Wu et al., 2019). Among the 14 GBS strains in the literature, except for one, Ib/ST10/eps/PI-1 + PI-2a strain, that possessed the tetM gene, all others were sensitive to tetracycline. Serotype Ib is typically defined by CC10/bca/PI-1 + PI-2a (Jones et al., 2003). In our study, all type Ib/ST10 isolates possessed the same characteristic protein (bca/PI-1 + PI-2a) and tetracycline sensitivity, consistent with existing literature. Thus, at least for Ib/ST10 GBS strains, tetracycline resistance is unlikely to be the driving force for clone emergence. We cannot disregard that clonal expansion may also be driven by other factors, for example, herd immunity or enhanced virulence of the clones that had undergone recombination. Studies elucidating the cause of driven clonal expansion or contributions to GBS hostpathogen interactions should be conducted (Flores et al., 2015;Teatero et al., 2015).
Our study has several limitations. Firstly, the study was conducted at a single center and the number of strains collected was relatively small. Additionally, most of our invasive GBS strains were type Ib/ST10, with other types having smaller case numbers. Further research enrolling multiple medical centers to obtain a more representative view of the Ib/ST10 GBS strain type is warranted. Secondly, we only employed PCR to study the microbiological characteristics of the GBS strains, as traditional targets lack the level of definition concerning genetic relatedness and pathogenic features. Going forward, a large-scale DNA sequencing approach to elucidate the population genetics of type Ib/ST10 GBS strains should be used.
In conclusion, we are the first to report the clinical and microbiological characteristics of type Ib/ST10 GBS strains isolated from infants with invasive GBS disease in China. Considering the probable clonal expansion and the emerging multi-drug resistant isolates, ongoing monitoring for type Ib/ ST10 GBS infections is warranted. Moreover, further studies focused on understanding novel adaptive traits and more aggressive treatments are of importance in the future.  Kang et al., 2017China (2002-2012 Invasive or colonizing infections 2/2.3 S Wu et al., 2017Portugal (2005 Invasive or colonizing infection 1/0.5 eps PI-1,PI-2a tetM Martins et al., 2017China (2008 Invasive

ETHICS STATEMENT
The study protocol was approved by the ethics committee of Shanxi Children's Hospital Shanxi Maternal and Child Health Hospital. This study required neither study-specific nor any other interventions and the data were analyzed anonymously. Therefore, written informed consent from the enrolled patients was waived by the ethics committee.