Group B Streptococcus CRISPR1 Typing of Maternal, Fetal, and Neonatal Infectious Disease Isolates Highlights the Importance of CC1 in In Utero Fetal Death

ABSTRACT We performed a descriptive analysis of group B Streptococcus (GBS) isolates responsible for maternal and fetal infectious diseases from 2004 to 2020 at the University Hospital of Tours, France. This represents 115 isolates, including 35 isolates responsible for early-onset disease (EOD), 48 isolates responsible for late-onset disease (LOD), and 32 isolates from maternal infections. Among the 32 isolates associated with maternal infection, 9 were isolated in the context of chorioamnionitis associated with in utero fetal death. Analysis of neonatal infection distribution over time highlighted the decrease in EOD since the early 2000s, while LOD incidence has remained relatively stable. All GBS isolates were analyzed by sequencing their CRISPR1 locus, which is an efficient way to determine the phylogenetic affiliation of strains, as it correlates with the lineages defined by multilocus sequence typing (MLST). Thus, the CRISPR1 typing method allowed us to assign a clonal complex (CC) to all isolates; among these isolates, CC17 was predominant (60/115, 52%), and the other main CCs, such as CC1 (19/115, 17%), CC10 (9/115, 8%), CC19 (8/115, 7%), and CC23 (15/115, 13%), were also identified. As expected, CC17 isolates (39/48, 81.3%) represented the majority of LOD isolates. Unexpectedly, we found mainly CC1 isolates (6/9) and no CC17 isolates that were responsible for in utero fetal death. Such a result highlights the possibility of a particular role of this CC in in utero infection, and further investigations should be conducted on a larger group of GBS isolated in a context of in utero fetal death. IMPORTANCE Group B Streptococcus is the leading bacterium responsible for maternal and neonatal infections worldwide, also involved in preterm birth, stillbirth, and fetal death. In this study, we determined the clonal complex of all GBS isolates responsible for neonatal diseases (early- and late-onset diseases) and maternal invasive infections, including chorioamnionitis associated with in utero fetal death. All GBS was isolated at the University Hospital of Tours from 2004 to 2020. We described the local group B Streptococcus epidemiology, which confirmed national and international data concerning neonatal disease incidence and clonal complex distribution. Indeed, neonatal diseases are mainly characterized by CC17 isolates, especially in late-onset disease. Interestingly, we identified mainly CC1 isolates responsible for in utero fetal death. CC1 could have a particular role in this context, and such a result should be confirmed on a larger group of GBS isolated from in utero fetal death.

General comments: Include the main findings and conclusions at the beginning and end of the discussion section. Explain why CRISPR typing was used instead of other typing methods and include the basic strengths and limitations of using this approach. How do your results differ from other results? Explain how the results were quality assured and whether QC strains were used for the study. Include the limitations sections because almost all studies have at least some of these.
Minor comments: Several abbreviations in the text are not explained: DR, , DRT, MGE etc.
Reviewer #2 (Comments for the Author): In this study, Brice and colleagues used the CRISPR1 locus sequencing method to characterize GBS isolates responsible for maternofetal infectious disease at the University Hospital of Tours, France, between 2004 and 2020. The authors assigned the GBS isolates to five different clonal complexes, mainly CC17 and CC1, by the CRISPR1 typing method. CC1 was the most common among GBS isolates associated with in utero death and the authors highlights the possibility of a particular role of this clonal complex in in-utero infection. Lastly, the authors suggest that isolates from in utero fetal death may differ from those responsible for EOD or LOD, and this should be investigated on a larger population. This is an interesting research article with clear goals and study parameters and procedures. However, there are some considerations to be addressed by the authors, as indicated below.
Overall, the manuscript should be revised regarding the language and the quality of figures should be improved. In addition, the manuscript should be revised regarding the CRISPR1 typing results and the corresponding conclusions shown by the authors in this paper. Although the experimental design of the study is acceptable, the manuscript does not clearly present robust and accurate data regarding CRISPR1 typing.
Abstract: Although the title of the manuscript highlights the importance of CRISPR1 typing of GBS strains associated with neonatal disease, CRISPR typing results are not addressed properly in the abstract. The authors include prevalence data of EOD and LOD overtime and the clonal complexes associated with neonatal disease cases, but at any moment the CRISPR1 typing method is cited or discussed.
Lines 106-108: please provide the number of GBS isolates recovered from each clinical source (blood culture, cerebrospinal fluid, or other sterile sites such as joint fluid or placenta) and origin (newborns, aborted or stillborn fetus, or pregnant women).
Lines 120-121: sequencing of PCR products was performed by Sanger sequencing technique? By using which equipment? Please include these methodological details in the text.
Lines 123-127: What was the rationale behind the analysis of CRISPR sequences? Which CRISPR features were considered in this analysis? Which dictionary of spacers was used? Please include in the text all these methodological details. In addition, the authors should describe in more detail the macro-enabled Excel tool used in the analysis of CRISPR sequences and provide a copy of the spreadsheet as a supplementary material.
Lines 159-161: Were the GBS isolates previously submitted to the MLST conventional analysis (sequencing the 7 housekeeping genes of the GBS scheme)? This result is crucial to validate the CRISPR1 typing analysis to assign clonal complexes shown by the authors in this study. The results of CRISPR1 typing method performed in this study must be compared to the results of CC assignment by the conventional MLST analysis for the GBS isolates analyzed in the study to validate the results obtained.
Lines 169-170: The results of single nucleotide polymorphism analysis of GBS isolates not distinguished by CRISPR1 typing method must be shown in the paper.
Staff Comments:

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Editor Spectrum
Dear Editor, I have thoroughly read the manuscript entitled: "Group B Streptococcus CRISPR1 typing of maternal, fetal and neonatal infectious disease isolates highlights the importance of CC1 in in utero fetal death ", and have given it my full consideration.

Summary:
The authors analyzed 115 GBS isolates with CRISPR typing. They found CC17 to be predominant overall and among isolates with late onset disease. They found several CC1 isolates among isolates with fetal death in utero. This is an interesting manuscript that defines the GBS population locally. The method used is not standardized and involves sequencing of individual loci and correlation with ST, but not actual ST determination by MLST or WGS.

General comments:
Include the main findings and conclusions at the beginning and end of the discussion section. Explain why CRISPR typing was used instead of other typing methods and include the basic strengths and limitations of using this approach. How do your results differ from other results? Explain how the results were quality assured and whether QC strains were used for the study. Include the limitations sections because almost all studies have at least some of these.

Minor comments:
Several abbreviations in the text are not explained: DR, , DRT, MGE etc.

Strengths:
Disclossing local epidemiology of GBS in all major disease types

Limitations:
Poor structure of the discussion Niche method used for typing

Reviewer #1 (Comments for the Author):
Summary: The authors analyzed 115 GBS isolates with CRISPR typing. They found CC17 to be predominant overall and among isolates with late onset disease. They found several CC1 isolates among isolates with fetal death in utero. This is an interesting manuscript that defines the GBS population locally. The method used is not standardized and involves sequencing of individual loci and correlation with ST, but not actual ST determination by MLST or WGS.
General comments: 1) Include the main findings and conclusions at the beginning and end of the discussion section.
We followed the reviewer's advice and included the main findings and conclusions at the beginning (lines 227-230 of the revised manuscript) and end of the discussion section (lines 315-322).
2) Explain why CRISPR typing was used instead of other typing methods and include the basic strengths and limitations of using this approach.
MLST is the reference typing method for GBS nowadays, but remains time-consuming and generates large volumes of data (7 sequences of nearly 500bp per isolate). With other teams, we participated in the development of the CRISPR typing method which presents a well-demonstrated discriminatory power and defines groups that perfectly match MLST-based clustering. Using single locus sequencing, this method offers a good compromise between discriminatory power, phylogenetic data and simplicity (Lopez-Sanchez et al., 2012, Lier et al., 2015, Beauruelle et al., 2017Beauruelle et al., 2018, Beauruelle et al., 2021. We modified the text in order to explain more precisely why CRISPR typing was used, including the basic strengths and limitations of this method (lines 236-248). Nevertheless, CRISPR typing is quite less discriminant for CC1/CC19 isolates. Thus we have determined SNP profile, a method avoiding a sequencing step, to complete typing. Results are presented extensively as supplementary data.
3) How do your results differ from other results?
Firstly, the aim of this study was to provide data regarding the local epidemiology (University hospital of Tours) of GBS strains responsible for invasive infections in mothers and newborns, including infections associated with in utero fetal death. The main clonal complex represented in newborn infections, especially in late-onset disease, was CC17, which was already known and described in several studies. Our data do not differ from other results regarding this conclusion, but we were able to show it using the CRISPR typing method which has rarely been used.
We were also able to show some interesting results concerning isolates responsible for infections associated with in utero fetal death. The main CC implicated was unexpectedly shown to be CC1, without any CC17 isolates, which was only reported in a recent Israeli study (Schindler et al. 2020) (lines 304-305). Actually, we had few isolates to analyze in our study, and this is why we suggest performing a typing study on a larger number of isolates responsible for in utero fetal death. This result is the main finding that we wanted to highlight, because CC1 is more frequently isolated in non-pregnant adult infections (Tsai et al. 2019) and not specifically known to be implicated in neonatal or in utero infections. This last reference was added in the revised version of the manuscript. 4) Explain how the results were quality assured and whether QC strains were used for the study.
We assured the quality of our results in two steps. First, as specified in the text, the PCR amplification was verified by electrophoretic migration in a 1% agarose gel (line 144-146). Then, concerning Sanger sequencing data, we used the ApE-A plasmid Editor v2.0.47© for analysis of sequences. The first and last parts of the sequence obtained were removed, and only the well-characterized sequence on the chromatogram with unique peaks was used for each isolate (lines 153-159).
Moreover, DNA sequences of the terminal direct repeat (TDR) and the spacers expected for each major clonal complex are known and have been published (Beauruelle et al., 2021). Their graphic representation is shown in the table 2. 5) Include the limitations sections because almost all studies have at least some of these.
We added a paragraph on limitations to the discussion section. The main ones are the monocentric aspect of the study, and the low number of isolates associated with in utero fetal death (lines 252-261).

Minor comments:
Several abbreviations in the text are not explained: DR, , DRT, MGE etc.
We corrected this oversight and took care to explain all abbreviations used in the text.

Reviewer #2 (Comments for the Author):
In this study, Brice and colleagues used the CRISPR1 locus sequencing method to characterize GBS isolates responsible for maternofetal infectious disease at the University Hospital of Tours, France, between 2004 and 2020. The authors assigned the GBS isolates to five different clonal complexes, mainly CC17 and CC1, by the CRISPR1 typing method. CC1 was the most common among GBS isolates associated with in utero death and the authors highlights the possibility of a particular role of this clonal complex in in-utero infection. Lastly, the authors suggest that isolates from in utero fetal death may differ from those responsible for EOD or LOD, and this should be investigated on a larger population. This is an interesting research article with clear goals and study parameters and procedures. However, there are some considerations to be addressed by the authors, as indicated below.
1) Overall, the manuscript should be revised regarding the language and the quality of figures should be improved.
The language was revised by asking a specialized organization, independent of our team, and we worked on improving the quality of figures.
2) In addition, the manuscript should be revised regarding the CRISPR1 typing results and the corresponding conclusions shown by the authors in this paper.
We revised our manuscript regarding the CRISPR1 typing results and the conclusions we made. Details are shown below.
3) Although the experimental design of the study is acceptable, the manuscript does not clearly present robust and accurate data regarding CRISPR1 typing.
We revised our manuscript in order to explain more precisely the robustness of CRISPR1 typing method. Details are shown below.
Abstract: Although the title of the manuscript highlights the importance of CRISPR1 typing of GBS strains associated with neonatal disease, CRISPR typing results are not addressed properly in the abstract. The authors include prevalence data of EOD and LOD overtime and the clonal complexes associated with neonatal disease cases, but at any moment the CRISPR1 typing method is cited or discussed.
We changed the abstract to provide further information concerning the use of the CRISPR1 typing method to obtain the CC affiliation of our isolates. (Lines 19-24).
Lines 106-108: please provide the number of GBS isolates recovered from each clinical source (blood culture, cerebrospinal fluid, or other sterile sites such as joint fluid or placenta) and origin (newborns, aborted or stillborn fetus, or pregnant women).
We added the number of GBS isolates recovered from each clinical source and origin (lines 121-123). Also, a supplementary data table summarizing all data of each isolate (origin, source, date of isola on, SNP result and CRISPR typing result with spacer representa ons) has been added. Addi onally, when working on the supplemental data table, a transcrip on error on one isolate was observed and fixed, and we apologize for that. This error had no impact on the conclusions we made (line 203 and figures 2 and 3).
Lines 120-121: sequencing of PCR products was performed by Sanger sequencing technique? By using which equipment? Please include these methodological details in the text.
We included these methodological details in the text (lines 147-150). We also provided details concerning the amplification protocol (lines 136-146) Lines 123-127: What was the rationale behind the analysis of CRISPR sequences?
As we have specified in the text (lines 83-88), CRISPR-Cas systems, by acquiring a part of DNA from MGE, reflect the different encounters between a bacterium and its environment. Thus, analysis of these different recent encounters can provide more discriminant power between strains even within a single clonal complex than MLST. On the other hand, ancestral spacers are conserved within clonal complexes defined by MLST, which explains the possibility of using this sequence analysis in epidemiological studies as an alternative to MLST.
Which CRISPR features were considered in this analysis?
Even if we were able to sequence the whole locus, only the terminal direct repeat and terminal spacers (one to four according to the clonal complex; table 2) were considered in this analysis, in order to group isolates into clonal complexes related to the MLST method. We have covered this in greater detail in the text (lines 185-195 and 236-241).
Which dictionary of spacers was used?
We used the dictionary of spacers made in the previous studies and published by Beauruelle et al., 2021. Please include in the text all these methodological details.
In addition, the authors should describe in more detail the macro-enabled Excel tool used in the analysis of CRISPR sequences and provide a copy of the spreadsheet as a supplementary material.
We have added further details of the tools used (lines 153-159) and the cleaned sequences, containing only spacers as graphic representations were added to the supplementary data.
Lines 159-161: Were the GBS isolates previously submitted to the MLST conventional analysis (sequencing the 7 housekeeping genes of the GBS scheme)? This result is crucial to validate the CRISPR1 typing analysis to assign clonal complexes shown by the authors in this study. The results of CRISPR1 typing method performed in this study must be compared to the results of CC assignment by the conventional MLST analysis for the GBS isolates analyzed in the study to validate the results obtained.
The GBS isolates analyzed by CRISPR1 typing in this work were not analyzed by the MLST method. Indeed, sequential studies had already been performed to validate the CRISPR1 typing as an alternative method for CC assignment. We added more details about this to the introduction section (lines 88-110). Over the years, this alternative method has provided many data correlated to MLST. Thus, for the present study, we considered CRISPR1 typing to be validated, and our objective was to use it instead of MLST, because of its conveniency, while also expanding the library of spacers.
Lines 169-170: The results of single nucleotide polymorphism analysis of GBS isolates not distinguished by CRISPR1 typing method must be shown in the paper.
We added the results of single nucleotide polymorphism analysis to supplementary data, which summarizes all isolate-related data (origin, source, date of isolation, SNP result and CRISPR typing result with spacer representations). Thank you for submitting your manuscript to Microbiology Spectrum. As you will see your paper is very close to acceptance. Please modify the manuscript along the lines recommended by a reviewer. As these revisions are quite minor, I expect that you should be able to turn in the revised paper in less than 30 days, if not sooner.
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Editor, Microbiology Spectrum Reviewer comments: Reviewer #1 (Comments for the Author): Much improved manuscript. Thank you for your explanations.
Reviewer #2 (Comments for the Author): The manuscript was extensively revised, and the authors addressed all comments accordingly, although there is a last suggestion to be made. Table 2 should include a legend for each graphical representation of each spacer and each DRT.

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