MLST Application in Analysis of Relationship between Group B Streptococcus Resistance Gene and Induction Resistance in Shanghai, China

Background With the gradual severe bacterial resistance and slow development of antibiotics, drug-resistant bacterial strains are widely distributed and have become a serious public health problem. Group B Streptococcus (GBS) which cause Group B strep-related disease is the major cause of severe infection in newborns. However, Clindamycin resistance of GBS induced by Erythromycin is emerging and become important clinical concerns today. A retrospective study was conducted on the drug resistance analysis of GBS strains isolated from Obstetrics and Gynecology Hospital from Jan 2016 to Dec 2017. The clinical and microbiological data including patient demographics, antimicrobial susceptibility testing, relative distribution drug resistance-associated genes mefA & ermB to Erythromycin, and multilocus sequence typing (MLST typing) were collected and analyzed. The Kirby-Bauer and VITEK2-compact were used to perform the susceptibility testing. The double disk diffusion method (D-test) was used for the detection of inducible clindamycin resistance. MLST was employed to identify sequence types of these strains. Polymerase Chain Reaction (PCR) was conducted to detect the drug resistance genes mefA & ermB to Erythromycin. resistance ratio of ST19 to Levofloxacin (75.0%) was higher than that of other ST types. The relevance resistance ratio of mefA and ermB was respectively 27.0% and 41.3% among 63 GBS isolates. Our study not only demonstrated a genetic diversity in iMLS B GBS in Shanghai through the analysis of MLST typing and resistance genes, but also found that there exist different distribution patterns of resistance and related resistance genes between different ST types. These findings would provide theoretical support for clinical prevention and treatment of resistant iMLS B GBS infection.


Introduction
Streptococcus agalactiae (Group B streptococcus; GBS) is the main cause of early-onset neonatal sepsis and meningitis, and is also an important cause of severe invasive infections in pregnant women, immunocompromised adults and the elderly around the world [1,2]. Importantly, GBS disease was also associated with significant morbidity and mortality, especially are the leading cause of life-threatening neonatal bacterial infections in developed countries [3,4]. Since then, the researches about GBS received extensive attention, and it was still under investigation as well in epidemiology aspects as in genetic background [5]. US Center for Disease Control and Prevention(CDC)respectively published and revised GBS prevention & treatment guidance in 1996, 2002 and 2010 [6][7][8]. In China, the GBS colonization rate of pregnant women were various in different regions, which was 8.2% in Guangdong, 4.2%-5.2% in Nanjing, 6.7% in Beijing and 9.0%-10.0% in Zhejiang Province and Shanghai [9][10][11].
In recent years, with GBS prenatal screening and intrapartum anti-GBS therapy of pregnant woman, especially in some developed regions, there has been a sharp decline in the proportion of neonatal GBS-induced bloodstream infections or serious complications in China [10,12]. However, due to the prevalence of multi-drug resistance of GBS, the resistance rate of erythromycin and clindamycin in GBS increased seriously [13,14].
Inducible type of MLS B resistance (iMLS B ) and cMLS B phenotypes were distinguished by Dtest as detailed by the Clinical & Laboratory Standards Institute (CLSI) [15]. According to the statistics collected from Grade III hospitals published by Chinese Antimicrobial Resistance Surveillance System in 2017, the drug resistance rate of GBS to Erythromycin and Clindamycin was over 60% and 50% respectively, which seriously affected the therapeutic effect in special groups who suffered from GBS infection. There are plenty of resistance gene types relating to GBS macrolide antibiotic resistance. Studies showed that the erythromycin ribosomal methylase encoded with ermB and macrolide antibiotic resistance induced by efflux pump encoded by mefA were popular in Asia [10].
In addition to the severe situation of drug resistance, GBS infection is bound to become a substantial risk to the obstetric diseases associated to pregnant women and newborn children [16]. Additionally, there were significant differences in drug resistance, molecular and serotype of GBS between diseases categories, specimen types or regions [17,18].
MLST of GBS isolates from different countries indicated that only limited numbers including ST1, ST10, ST17, ST19 and ST23 were associated with colonizing or invasive isolates [19]. Among these STs, ST17 is a hypervirulent clone, mainly associated with invasive diseases in newborns, whereas ST19 can cause invasive diseases in both newborns and adults [20,21]. According to MLST analysis, the majority of the invasive GBS diseases in infants from urban areas in southern China belong to the genotype ST17 or ST17-like genotype [22]. cMLS B resistance (46%) mediated by the ermB gene was significantly associated with the Guelma isolates belonged to ST23 in France [23]. The molecular epidemiological study of GBS in Ireland showed that the isolates could be divided into 5 main clonal cluster (CC), among which CC1, CC17 and CC23 accounted for 67.2% [24]. In Iceland, the decline of serotype III was reflected in a decrease of clonal complexes CC17 and CC19 that included most serotype III isolates. On the other hand, the increase in frequency of CC1 was caused by ST1 and ST196 [25].
The study on multiple-resistant isolates can provide guidance in the case of treatment failure to complication induced by early-onset GBS infection through utilizing prophylactic antibiotic [26,27]. To date, the research of iMLS B GBS, which was of great significance for the prevention of infectious diseases and the treatment of severe complications to childbearing age women and newborn babies, is still limited in China. Moreover, the analysis of GBS resistance also showed a great necessity to the treatment for recurrent episodes of gynecologic inflammation and spontaneous abortion [28,29]. As one of famous gynecologic & obstetrics hospitals in Asia and the world, Obstetrics and Gynecology Hospital affiliated to Fudan University has a larger proportion of high-risk pregnant women all over the country. In this study, the clinical features, drug resistance, drug resistance gene and MLST of iMLS B GBS were analyzed, which would provide basic data for clinical prevention and treatment of GBS resistance. isolates were cultured for GBS on blood agar plates with 5% sheep blood 24 hours at 37 ℃. For neonatal patients, the isolates were originated from the blood culture specimen.
The isolated colonies after 24 hours culture was identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Bruker Daltonics Inc, Massachusetts, USA). In our study, all the strains' information can be timely identified during or after data collection. This study was performed in accordance with human subject protocols approved by the Ethics Committee (Ethics Approval Number: KYY-2016-22) of Obstetrics and Gynecology Hospital of Fudan University.

Inhibitory Concentration
For the antimicrobial susceptibility characterization, VITEK2-compact full-automatic bacterial susceptibility instrument and relevant bacterial susceptibility identification cards (bioMérieux Corporate in France) were used for detection and antibiotic susceptibility testing [30]. Streptococcus pneumoniae ATCC49619 was used as the reference strain.
Inducible type of MLS B resistance (iMLS B ) and cMLS B phenotypes were distinguished by Dtest with Mueller-Hinton agar with 5% of sheep blood (Shanghai Kehua Biological Co., Ltd., China) as detailed by CLSI. The susceptibility results were interpreted and analyzed based on M100-S27 document of CLSI breakpoints.

Multilocus Sequence Typing
Furthermore, for serotype and genotype determination, all GBS positive cultures were genetically characterized by MLST as described previously [31]. Briefly, the bacterial isolates were grown in their preferred medium and pelleted. 7 housekeeping genes of adhp,pheS atr glnA sdhA glck tkt in GBS were amplified by PCR [32]. The amplification system and sequence primers were determined by MLST (http://www.mlst.net/). The total volume of reaction system was 25 µL, including 17 µL of ddH 2 O, 2.5 µL of 10 × PCR buffer, 0.5 µL of dNTP, 0.25 µL of Taq DNA Polymerase, 1.5 µL of magnesium ion (25 mM), 1 µL for upstream primer and downstream primer each and 1.25 µL of template DNA. The loop condition: pre-denaturation at 94℃ for 5 min, denaturation at 94℃ for 30 sec, annealing at 56℃ for 30 sec, extension at 72℃ for 1 min, and 30 cycles in total. The amplification product was analyzed by 1% of agarose gel electrophoresis. DNA extraction kit was purchased from Sangon Biotech (Shanghai) Co., Ltd. and the sequencing was performed by Beijing Genomics Institute (BGI, Beijing) Co., Ltd. The results were compared with the MLST databases online (http://pubmlst.org/sagalactiae/) to determine genotypes and ST types.

Determination of antimicrobial resistance gene markers by PCR
To investigate the drug resistance associated genes, PCR was carried out to detect the drug resistance genes mefA and ermB to GBS strains (positive to D-inhibition zone trial).

Statistical Analysis
The results of antimicrobial susceptibility testing were analyzed by WHONET5.6 statistical software. BioNumerics 7.5(Applied Maths, Belgium)analysis was conducted to investigate the affinity of these isolates. Minimal Spanning Trees is a subset of the edges of a connected, edge-weighted directed graph that connects all the vertices together. Data were presented as mean values with SD or median with interquartile range when appropriate. Statistical significance was defined at a P value of < 0.05.

Patient demographic characteristics
In total, 31894 patients were investigated from Obstetrics and Gynecology Hospital affiliated to Fudan University between Jan, 2016 and Dec, 2017. 1021 strains of GBS were compiled in Table 1 (Positive detection rate

Multilocus Sequence Typing Analysis
All 63 iMLS B GBS strains were typed by MLST (in Fig. 1

Resistance patterns against various antimicrobial agents
For the resistance patterns of these 63 iMLSB GBS strains, none of the 63 iMLSB GBS were resistant to penicillin, Vancomycin, Linezolid, Clindamycin and Nitrofurantoin.
Intermediate rate of iMLS B GBS to Nitrofurantoin was 9.5%. As shown in Fig. 2  It was much higher than the rate in Brazil (20%) and Africa (17.4%) [44,46].
Understanding the distribution of GBS infection and drug resistance is of great significance for guiding rational drug use in clinical practice, especially for the rational drug use of iMLSB GBS strain. Therefore, it is imperative to strengthen the monitoring of to inflammation of pregnancy women in Obstetrics was CC19. The highest incidence of cervical inflammation in Gynecology was CC19 and adverse pregnancy outcomes in Birth Control Department was ST12. In other states and regions, CC19, CC1, CC10 and CC17 were the most in Romania from Eastern Europe and CC23, CC19 and CC17 were mainly detected in Poland from Central Europe [47,48]. Like Iran, the most common clonal complexes were CC19 and CC10 [39,49]. CC17 was the main GBS strains to induce invasive diseases of newborn babies in Beijing, China [11]. At the same time, CC17 was predominant in GBS neonatal infections in France [42,50]. CC17 strains cause both neonatal and adult invasive infections which cluster tightly in a phylogenetic tree, signifying that they are derived from the same genetic pool in Canada [51]. The research to capsular polysaccharide on virulent strain's surface had important implications for the development of GBS vaccine [52].
Multiple resistance isolates severely threaten the public health due to the pressure selection of antibiotics. Among 63 iMLSB GBS isolates, the drug resistance rate of ST19 to Levofloxacin (75.0%) was higher than that of other types, while ST12 was susceptible to Levofloxacin. Similarly, the predominant genotype of the levofloxacin-resistant isolates was ST19 in Taiwan [53]. This rate was close to that of ST19 from non-genitourinary tract specimens reported in Shanghai (76.9%). and lower than III/ ST19 resistance to Levofloxacin in Beijing (> 90%) [54]. What's different is that ST19 was reported as the isolates with lower susceptibility to Penicillin in Japan [55]. Those strains not susceptible to Penicillin were usually resistant to macrolides and fluoroquinolone antibiotics. The drug resistance rate of CC10 to Levofloxacin (7.69%) was obviously lower than Korean [56]. We found the distinct gaps and genetic diversity exist in the drug resistance and relevant resistance genes with different ST types. These data suggested that the epidemiological investigation of GBS MLST in Shanghai has important clinical reference.
Resistance to erythromycin in neonatal invasive GBS has been reported worldwide, and the resistance to erythromycin from GBS strains was mostly mediated by ermB, ermA and The relevance ratio of mefA and ermB was 27.0% and 41.3% respectively in this study and only 4 strains included the both genetypes, which was close to ermB relevance ratio (47.1%) and largely different to mefA relevance ratio (0%) for Erythromycin resistant GBS strains separated from urine specimen in Suzhou, China [57,58]. In general, Erythromycin resistance induced by efflux pump was negative in D-test. However, 27% of induced resistant strains included mefA and was mostly concentrated on CC19 in this research.
These isolates might spontaneously mutate from induced resistance to structural resistance and continue to show high resistance to Erythromycin. Treatment with clindamycin may induce antibiotic strain resistance, even leading to treatment failure.
Therefore, the further study of CC19 spontaneously-mutated isolates could be regarded as the important supplement to GBS resistance system and gave the guidance on rational use of antibiotics for clinicians to avoid the occurrence of drug resistance.
In general, Erythromycin resistance induced by efflux pump was negative in D-test.

Consent for publication
Consent to publish is obtained from all the individuals whose data contained in this manuscript.

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Authors' contributions
Jing Gao composed the manuscript and analyzed the data. Yisheng Chen,Yiqian Peng, Nanyan Jiang and Ying Zhang provided the samples and collected the clinical data.
Chunmei Ying designed and coordinated the study. All the authors read and approved the final manuscript.
We would like to thank Mingquan Guo from Department of Laboratory Medicine, Shanghai Public Health Clinical Center for the help with critical review of the article.