Whole genome sequencing of Neisseria meningitidis Y isolates collected in the Czech Republic in 1993-2018.

INTRODUCTION: The study presents the analysis of whole genome sequencing (WGS) data for Neisseria meningitidis serogroup Y isolates collected in the Czech Republic and their comparison to other countries. The aim of the study was to determine whether there are lineages of N. meningitidis serogroup Y in the Czech Republic genetically related to foreign ones that have been causing an increase of the morbidity and the mortality of invasive meningococcal disease (IMD) world-wide recently.
MATERIAL AND METHODS: The WGS data of 43 Czech N. meningitidis Y isolates, 35 from IMD and 8 from healthy carriers were analysed. Due to the potential of meningococcal B vaccines to induce protection against non-B serogroups, the coverage of Czech isolates of N. meningitidis Y by these vaccines was studied. The WGS data of Czech, European and non-European isolates of N. meningitidis serogroup Y were compared.
RESULTS: WGS assigned 36 isolates of N. meningitidis Y to five clonal complexes: cc23, cc92, cc167, cc103, and cc174, while seven isolates remained unassigned to any clonal complexes (ccUA). Eighteen invasive isolates belonged to clonal complex cc23, which was detected throughout the studied years. The occurrence of cc23 was recorded in all age groups of IMD patients, with the highest found in those aged 15-19 years. On the phylogenetic network isolates of cc23 form a separate lineage, distinct from all other isolates of N. meningitidis Y. The remaining isolates were assigned to other clonal complexes and have very low relatedness to cc23 isolates and to each other. The comparison with foreign WGS data showed that within the main genetic lineages, which are defined by clonal complexes, Czech isolates of N. meningitidis Y, similar to European ones, mostly cluster together and form geographical sublineages.
CONCLUSIONS: WGS analysis showed the population of Czech N. meningitidis Y isolates as relatively heterogeneous, containing a large number of genetic lineages. The Czech isolates of N. meningitidis Y follow the trend observed for European isolates. Our result was one of the bases for updating the recommended vaccination strategy in the Czech Republic.

Introduction

Invasive meningococcal disease (IMD) is one of the most severe infectious diseases worldwide with a high fatality rate and a high risk of life-long disabling sequelae in survivors [1-3]. IMD is caused by the bacterium Neisseria meningitidis, detected in the upper respiratory tract of up to 10% of the healthy population, referred to as healthy carriers of meningococci. The serogroups of N. meningitidis most often involved in IMD are A, B, C, X, W, and Y. Their distribution varies between countries [4-6].

The incidence of IMD and causative serogroups vary over time partly due to a combination of effects of vaccination programmes and also natural fluctuation. After meningococcal C conjugate vaccines were introduced in the early 2000s, a sharp decline in IMD caused by N. meningitidis serogroup C was observed in a number of countries. When meningococcal A conjugate vaccine became available in the African meningitis belt it practically led to the elimination of IMD caused by N. meningitidis serogroup A in this area [3]. Consequently, other causative serogroups started to be dominant. Increasing trends were observed particularly for serogroups W and Y, which resulted in switching from monovalent meningococcal C conjugate vaccines to quadrivalent meningococcal A, C, W, Y conjugate vaccines in many countries [3].

Since the 1990s IMD caused by serogroup Y showed an upward trend and increased case fatality rate in the USA, Canada, Latin American countries, but also in Europe, particularly in Sweden [6-14]. Before the worldwide rise in IMD caused by serogroup Y, this serogroup was detected primarily in healthy carriers, being rarely the cause of IMD and if so, particularly in the elderly population. The recent rise in IMD cases caused by serogroup Y has also been reported in younger age groups [13]. Molecular characterization of isolates of N. meningitidis Y has shown that the recent rise in IMD caused by this serogroup is primarily linked to clonal complex cc23, which, however, had a relatively low invasive potential compared to other hyperinvasive lineages [14].

In the Czech Republic, data on the incidence of IMD have been available since 1943 (passive reporting) along with surveillance data since 1993 [15]. The surveillance data-based analysis of the epidemiological situation of IMD in the Czech Republic and the meningococcal vaccination guidelines are published annually in the Bulletin of the Centre for Epidemiology and Microbiology (http://www.szu.cz/publikace/zpravy-epidemiologie-a-mikrobiologie). The IMD surveillance data in the period from 1993 to 2018 show the highest incidence 2.2/100 000 population in 1995. After this, the incidence gradually decreased reaching the minimum 0.4/100 000 population in 2014 and 2016. The IMD surveillance data from 1993 to 2018 indicate that serogroups B and C are dominant in the Czech Republic (ranging from 20.4 to 71.9% and from 6.6 to 59.2%, respectively), but since the early millennium, serogroups W and Y have been on the rise: up to 9.3% and 5.9%, respectively (S1 Fig). The IMD surveillance programme in the Czech Republic also includes molecular characterization of isolates performed by the National Reference Laboratory for Meningococcal Infections (NRL) [15], which has been extended by the WGS method since 2015 [16-18].

Several polysaccharide conjugate vaccines against serogroups A, C, W and Y are available [19]. Two recombinant vaccines including serogroup B meningococcal antigens (MenB vaccines) have been developed: MenB-4C vaccine (Bexsero) and MenB-fHbp vaccine (Trumenba) [20, 21]. Genes encoding MenB vaccine antigens are also present in strains belonging to other meningococcal serogroups and for this reason, theoretical coverage of non-B N. meningitidis isolates by MenB vaccines is being studied worldwide [22-25]. Protection by MenB vaccines against non-B N. meningitidis represents added value in vaccination programs [26]. Vaccines registered by the European Medicines Agency (EMA) are available in the Czech Republic for vaccination against IMD–two quadrivalent conjugate vaccines: MenACWY-TT vaccine (Nimenrix) and MenACWY-CRM vaccine (Menveo) and both MenB vaccines (Bexsero, Trumenba). To achieve the most comprehensive possible immunity against IMD, the combination of a quadrivalent ACWY conjugate vaccine and MenB vaccine is recommended in the Czech Republic. In accordance with Czech legislation, vaccination against IMD is covered by health insurance for persons with underlying diseases (since January 2018) and small children (since May 2020).

Since 1993, the overall case fatality rate of IMD in the Czech Republic has been ranging from 4.7 to 16.4%, averaging at 10.2%. The highest case fatality rate is due to IMD caused by N. meningitidis Y averaging at 15.5%. Molecular characterization of N. meningitidis Y isolates provides the necessary background data for setting the vaccination strategy in the Czech Republic. We present results of the WGS analysis of 43 Czech isolates of N. meningitidis Y from 1993–2018 and their comparison with the WGS data from other countries. The aim of the study was to determine whether there are lineages of N. meningitidis serogroup Y in the Czech Republic genetically related to foreign ones that have been causing an increase of the morbidity and the mortality of invasive meningococcal disease world-wide recently.

Material and methods

Neisseria meningitidis isolates

Isolates of N. meningitidis from IMD patients are referred to the NRL by microbiological laboratories from all over the Czech Republic for confirmation and further characterization in accordance with the Czech legislation. The isolates from healthy carriers are referred to the NRL voluntarily and in small numbers. All isolates received are stored freeze-dried and/or frozen (-80°C, Cryobank B, ITEST) in the NRL collection. For each isolate, clinical, epidemiological, and microbiological data are available in the electronic database of the NRL collection. A total of 43 N. meningitidis serogroup Y isolates from 1993–2018 were studied, 35 from IMD patients and 8 from healthy carriers, to determine if there is a difference in isolates from patients with IMD and from healthy carriers.

Characterization of Neisseria meningitidis

N. meningitidis isolates were cultured on Mueller Hinton chocolate agar at 37°C and 5% CO2 for 18–24 hours. The identification of N. meningitidis was confirmed using the API NH kit (BIOMÉRIEUX). Serogroups were determined by conventional serological methods (Pastorex Meningitis Bio-RAD, antisera N. meningitidis ITEST, Bio-RAD) and confirmed by RT-PCR.

Whole genome sequencing and analysis

The methods used in the present study have been described in detail previously [17, 18]. DNA isolation was performed using the QIAamp DNA Mini Kit (QIAGEN) according to the manufacturer’s instructions. DNA was sent for WGS to the European Molecular Biology Laboratory, Heidelberg, Germany (EMBL). The Illumina MiSeq platform was used for sequencing and the result was overlapping sequences approximately 300 bp in length. The genome assembly from primary raw WGS data was performed using the Velvet de novo Assembler software with Velvet-Optimiser script [27]. Isolate genome assemblies were submitted to the PubMLST Neisseria database (https://pubmlst.org/) [28, 29] and automatically characterized by the BIGSdb platform at finetyping loci (porA, fetA) [30], MLST genes (abcZ, adk, aroE, fumC, gdh, pdhC, and pgm) [31], MenB vaccine antigen genes (nhba, nadA, and fHbp) [32] and 53 ribosomal protein genes (rpsA–rpsU, rplA–rplF, rplI–rplX, rpmA–rpmJ) [33]. New gene and peptide variants were scanned manually, and after curator approval and annotation, were added to the PubMLST database. Genomes were compared using the BIGSdb Genome Comparator tool with the “all loci” scheme, providing the highest resolution. Incomplete loci (due to contig breaks) were ignored in pairwise comparisons in the distance matrix calculations. The distance matrices, which are based on the number and allelic variability of the genes contained in individual schemes, were generated automatically and phylogenetic networks constructed using the SplitsTree4 software using the NeighborNet algorithm [34]. Results were graphically edited by the Inkscape tool (www.inkscape.org/en/).

Isolates were assigned to sequence type (ST), clonal complex (cc) and ribosomal sequence type (rST) based on the allelic profile of MLST and ribosomal protein genes. Due to the potential of MenB vaccines to induce protection against non-B serogroups, the coverage of isolates by these vaccines was studied. The Bexsero Antigen Sequence Type (BAST) was determined from the combination of peptide variants of two variable regions of the PorA protein (VR1 and VR2) and peptide variants of three MenB-4C vaccine antigens (NHBA, NadA, and FHbp) [35]. From this, the theoretical coverage of a given isolate by both MenB vaccines (Bexsero and Trumenba) was determined using the Meningococcal Deduced Vaccine Antigen Reactivity (MenDeVAR) Index, recently introduced on the PubMLST database. The MenDeVAR index is based on analysis of the presence of particular peptide variants of MenB vaccine antigens, their expression, and bactericidal susceptibility test result [36].

The phylogenetic networks were created for invasive isolates of N. meningitidis Y from the Czech Republic (n = 35) together with European (n = 960) and non-European countries (n = 375), which were available in the PubMLST database by May 26, 2021 and met the following criteria: serogroup Y, genogroup Y, invasiveness, complete MLST profile, and sequencing data size of > 1.8 MB.

Results

Czech isolates of N. meningitidis Y

WGS assigned 36 of 43 isolates of N. meningitidis Y to five clonal complexes: cc23, cc92, cc167, cc103, and cc174, while seven isolates remained unassigned to any of the described clonal complexes (ccUA) (Table 1). Most prevalent was cc23 with 21 isolates (ST-23, n = 10; ST-569, n = 4; ST-1625, n = 6; and ST-8526, n = 1). Followed by cc92 with 7 isolates (ST-92, n = 6, and ST-94, n = 1), and cc167 with 6 isolates (ST-168), as well as one isolate each for cc103 (ST-11017) and cc174 (ST-1466). The most frequent clonal complex in isolates from IMD was cc23, followed by cc167, while in isolates from healthy carriers, cc92 followed by cc23 was most frequently found. The highest occurrence of IMD caused by N. meningitidis Y in the Czech Republic was recorded in adolescents/young adults (age 15–19), followed by older adults (65+ years of age) and in younger children (1–4 years old) (S2 Fig).

Table 1

Basic molecular characterization of N. meningitidis Y isolates collected in the Czech Republic from 1993 to 2018, (n = 43).

Strain IDs	PubMLST IDs	ST	abcZ	adk	aroE	fumC	gdh	pdhC	pgm	cc	porA VR1	porA VR2	fetA VR	rST
0059/93	966	92	3	7	4	37	8	18	21	ST-92	5–1	10–14	F5-14	3142
0111/93	1580	130	23	5	9	3	13	32	8	UA	18–7	9	F5-13	3297
0224/93	1614	94	3	7	4	35	8	18	2	ST-92	5–1	10–4	F1-5	3214
0323/93	8142	92	3	7	4	37	8	18	21	ST-92	5–1	10–1	F5-14	3006
0338/93	8152	92	3	7	4	37	8	18	21	ST-92	5–1	10–1	F5-14	3006
0200/94	83899	92	3	7	4	37	8	18	21	ST-92	5–1	10–4	F1-5	137524
0040/95	83871	569	10	5	93	14	11	9	17	ST-23	5–1	2–2	F5-8	2422
0449/95	83903	92	3	7	4	37	8	18	21	ST-92	5–1	10–4	F1-5	137524
0008/96	83865	130	23	5	9	3	13	32	8	UA	12–1	16–8	F5-13	137518
0264/99	83901	1625	10	5	18	14	11	9	17	ST-23	5–1	2–2	F5-8	137525
0172/01	83895	1625	10	5	18	14	11	9	17	ST-23	5–1	2–2	F5-8	2422
0179/01	83897	569	10	5	93	14	11	9	17	ST-23	5–1	2–2	F5-8	137523
0251/01	83900	23	10	5	18	9	11	9	17	ST-23	5–2	10–1	F4-1	2421
0055/02	83874	23	10	5	18	9	11	9	17	ST-23	7–2	13–2	F4-17	137520
0068/02	83877	1625	10	5	18	14	11	9	17	ST-23	5–1	2–2	F5-8	137521
0177/02	83896	569	10	5	93	14	11	9	17	ST-23	5–1	2–2	F5-8	2422
0066/03	83911	130	23	5	9	3	13	32	8	UA	18–12	10–2	F2-7	137529
0157/03	83922	3015	219	5	275	17	11	8	21	UA	5–2	10–1	F5-8	7234
0192/04	83898	1625	10	5	18	14	11	9	17	ST-23	5–1	2–2	F5-8	2422
0267/04	83902	569	10	5	93	14	11	9	17	ST-23	5–1	2–2	F5-8	2589
0065/05	83910	3015	219	5	275	17	11	8	21	UA	5–2	10–1	F5-8	137528
0075/05	83913	1625	10	5	18	14	11	9	17	ST-23	5–1	2–2	F5-8	2422
0094/07	83915	23	10	5	18	9	11	9	17	ST-23	5–2	10–1	F4-1	137531
0105/07	83917	130	23	5	9	3	13	32	8	UA	18–12	10–2	F2-7	137532
0156/07	83921	3015	219	5	275	17	11	8	21	UA	18–1	16–4	F3-4	137534
0190/07	83923	168	2	16	6	17	9	18	8	ST-167	5–1	10–4	F4-1	7517
0121/08	83920	23	10	5	18	9	11	9	17	ST-23	5–2	10–1	F4-1	2421
0125/08	106031	23	10	5	18	9	11	9	17	ST-23	5–2	10–1	F4-1	2421
0126/08	106032	23	10	5	18	9	11	9	17	ST-23	5–2	10–1	F4-1	2421
0049/09	83872	23	10	5	18	9	11	9	17	ST-23	5–2	10–1	F4-1	2421
0039/10	83870	168	2	16	6	17	9	18	8	ST-167	5–1	10–4	F4-1	7517
0063/10	83876	8526	10	344	18	9	11	9	17	ST-23	5–2	10–1	F5-12	7645
0004/11	83864	23	10	5	18	9	11	9	17	ST-23	5–2	10–1	F4-1	2421
0014/11	83904	1625	10	5	18	14	11	9	17	ST-23	5–1	2–2	F5-8	2422
0102/11	83916	168	2	16	6	17	9	18	8	ST-167	5–1	10–4	F4-1	7517
0089/12	83880	168	2	16	6	17	9	18	8	ST-167	5–1	10–4	F4-1	7517
0032/13	83868	23	10	5	18	9	11	9	17	ST-23	5–2	10–1	F4-1	2421
0042/13	36323	23	10	5	18	9	11	9	17	ST-23	5–2	10–1	F5-12	89807
0060/14	83875	11017	8	5	6	17	8	18	8	ST-103	7–2	13–2	F3-9	169886
0024/16	83867	168	2	16	6	17	9	18	8	ST-167	5–1	10–4	F4-1	125578
0019/17	83866	168	2	16	6	17	9	18	8	ST-167	5–1	10–4	-	7517
0037/18	101296	92	3	7	4	37	8	18	21	ST-92	5–1	10–4	F1-5	163667
0061/18	82068	1466	6	5	173	13	5	24	17	ST-174	21	16	F3-7	80732

Year of isolation is indicated in the strain ID; strains isolated from IMD are indicated in bold, isolates from carriers in normal font; VR1, VR2 = PorA variable region 1 and 2; VR = FetA variable region; ST = sequence type; cc = clonal complex, UA = clonal complex unassigned; yellow highlight = newly described allele, sequence type, or ribosomal sequence type.

Given the type of construction of the peptide MenB vaccines, among other benefits, they have the potential to provide protection against other meningococcal serogroups. According to the MenDeVAR index, the isolates are classified into four groups in relation to both MenB vaccines. Isolates containing one or more specific antigenic variants included in MenB vaccines are highlighted in green. Isolates containing one or more antigenic variants that showed cross-reactivity in experimental studies are highlighted in orange. Isolates for which enough data have not been available on their antigenic variants are highlighted in grey, and those carrying antigenic variants that did not show cross-reactivity in experimental studies are highlighted in red (https://pubmlst.org/organisms/neisseria-spp/mendevar). In the study collection of MenY isolates, phenomenon of potential protection can be observed particularly for the Trumenba vaccine (Table 2). The MenDeVAR index classified 32 of 43 isolates (nearly 75%) in the PubMLST database as covered by cross-reactivity. Experimental data are not yet available for the remaining 11 isolates. Regarding the Bexsero vaccine one isolate carried NadA peptide variant 8, which is included in this vaccine. One isolate appeared to be covered due to cross-reactivity and one isolate was considered as uncovered by the Bexsero vaccine based on the MenDeVAR index. For the remaining 40 isolates, experimental data for at least one antigenic variant was not available, and thus they could not be categorized based on the MenDeVAR index.

Table 2

Molecular characterization focused on MenB vaccine antigens of N. meningitidis Y isolates collected in the Czech Republic from 1993 to 2018, (n = 43).

Strain IDs	porA VR1	porA VR2	nhba allele	nhba peptide	nadA allele	nadA variant	nadA peptide	fHbp allele	fHbp peptide	fHbp variant	fHbp family	BAST type	Men DeVAR (Bexsero)	Men DeVAR (Trumenba)
0059/93	5–1	10–14	7	9	-	-	-	687	101	2	A	2917
0111/93	18–7	9	15	24	-	-	-	688	34	2	A	1479
0224/93	5–1	10–4	7	9	-	-	-	21	21	2	A	549	-	fHbp 21
0323/93	5–1	10–1	7	9	-	-	-	687	101	2	A	987
0338/93	5–1	10–1	7	9	-	-	-	687	101	2	A	987
0200/94	5–1	10–4	7	9	-	-	-	691	585	2	A	1501
0040/95	5–1	2–2	13	8	-	-	-	25	25	2	A	227		fHbp 25
0449/95	5–1	10–4	7	9	-	-	-	691	585	2	A	1501
0008/96	12–1	16–8	15	24	-	-	-	302	245	1	B	2987
0264/99	5–1	2–2	13	8	-	-	-	25	25	2	A	227		fHbp 25
0172/01	5–1	2–2	13	8	-	-	-	25	25	2	A	227		fHbp 25
0179/01	5–1	2–2	13	8	-	-	-	25	25	2	A	227		fHbp 25
0251/01	5–2	10–1	10	7	-	-	-	25	25	2	A	228		fHbp 25
0055/02	7–2	13–2	335	145	-	-	-	21	21	2	A	2988		fHbp 21
0068/02	5–1	2–2	13	8	-	-	-	25	25	2	A	227		fHbp 25
0177/02	5–1	2–2	13	8	-	-	-	25	25	2	A	227		fHbp 25
0066/03	18–12	10–2	15	24	-	-	-	302	245	1	B	1298
0157/03	5–2	10–1	3	20	-	-	-	16	16	2	A	1418		fHbp 16
0192/04	5–1	2–2	13	8	-	-	-	25	25	2	A	227		fHbp 25
0267/04	5–1	2–2	13	8	-	-	-	25	25	2	A	227		fHbp 25
0065/05	5–2	10–1	3	20	-	-	-	16	16	2	A	1418		fHbp 16
0075/05	5–1	2–2	13	8	-	-	-	25	25	2	A	227		fHbp 25
0094/07	5–2	10–1	10	7	-	-	-	25	25	2	A	228		fHbp 25
0105/07	18–12	10–2	15	24	-	-	-	1079	245	1	B	1298
0156/07	18–1	16–4	3	20	-	-	-	14	14	1	B	2990	fHbp 14	fHbp 14
0190/07	5–1	10–4	509	9	-	-	-	23	23	2	A	384		fHbp 23
0121/08	5–2	10–1	10	7	-	-	-	25	25	2	A	228		fHbp 25
0125/08	5–2	10–1	10	7	-	-	-	25	25	2	A	228		fHbp 25
0126/08	5–2	10–1	10	7	-	-	-	25	25	2	A	228		fHbp 25
0049/09	5–2	10–1	10	7	-	-	-	25	25	2	A	228		fHbp 25
0039/10	5–1	10–4	509	9	-	-	-	23	23	2	A	384		fHbp 23
0063/10	5–2	10–1	10	7	-	-	-	25	25	2	A	228		fHbp 25
0004/11	5–2	10–1	10	7	-	-	-	25	25	2	A	228		fHbp 25
0014/11	5–1	2–2	13	8	-	-	-	25	25	2	A	227		fHbp 25
0102/11	5–1	10–4	509	9	-	-	-	23	23	2	A	384		fHbp 23
0089/12	5–1	10–4	509	9	-	-	-	23	23	2	A	384		fHbp 23
0032/13	5–2	10–1	10	7	-	-	-	25	25	2	A	228		fHbp 25
0042/13	5–2	10–1	10	7	-	-	-	25	25	2	A	228		fHbp 25
0060/14	7–2	13–2	15	24	109	4/5	21	785	649	1	B	2972
0024/16	5–1	10–4	509	9	-	-	-	23	23	2	A	384		fHbp 23
0019/17	5–1	10–4	509	9	-	-	-	23	23	2	A	384		fHbp 23
0037/18	5–1	10–4	7	9	-	-	-	691	585	2	A	1501
0061/18	21	16	9	6	80	2/3	8	21	21	2	A	14	nadA 8	fHbp 21

Year of isolation is indicated in the strain ID; strains isolated from IMD are indicated in bold, isolates from carriers in normal font; VR1, VR2 = PorA variable region 1 and 2; yellow highlight = newly described allele or the BAST type; MenDeVAR: green, orange, grey, and red highlight = graphical representation of four groups with different functional effects in relation to both MenB vaccines.

Eighteen out of 35 invasive isolates belonged to clonal complex cc23, which was detected throughout the studied years, but showed no increase in any period (S3 Fig). The phylogenetic network (Fig 1) illustrates the relatedness of all study isolates of N. meningitidis Y from the Czech Republic. The genetic relationships between cc23 isolates are shown in more detail in a separate phylogenetic network (Fig 2).

Fig 1

Genetic relationship of N. meningitidis Y isolates collected in the Czech Republic from 1993 to 2018.

Neighbour-net network of MenY isolates (n = 43). Isolates are coloured according to detection year and labelled by their NRL number, ST, and cc. Invasive isolates are marked with a square, carriage isolates with a circle.

Fig 2

Genetic relationship of N. meningitidis Y isolates collected in the Czech Republic from 1993 to 2018, clonal complex cc23.

Neighbour-net network of MenY isolates cc23 (n = 21). Isolates are coloured according to detection year and labelled by their NRL number, ST, and cc. Invasive isolates are marked with a square, carriage isolates with a circle.

The most common clonal complex in our study set was cc23, which is represented by eighteen invasive and three carriage isolates. Isolates of cc23 form a separate lineage, distinct from all other isolates of N. meningitidis Y. The cc23 lineage splits into two sister sublineages, ST-23 and ST-569/1625. Eight of 10 ST-23 isolates form a highly compact cluster of sublineage ST-23. Sequence type ST-8526 (63/10) differs from ST-23 in the MLST genes by allele 344 of the adk gene, which was newly described in isolate 63/10 as well as ST-8526 itself. The other sublineage is formed by two genetically related clusters, ST-569 and ST-1625, which include almost exclusively invasive isolates of the respective sequence types. Interestingly, invasive isolate 55/02—Y: P1.7–2,13–2: F4-17: ST-23 (cc23), despite its standard MLST gene alleles, is the only cc23 outlier from sublineages ST-23 and ST-569/1625 (Fig 1). A more detailed phylogenetic analysis of cc23 isolates (Fig 2) showed that it has a unique genetic profile and is unrelated to any of the major sublineages of clonal complex cc23. In this isolate, a new ribosomal sequence type, rST-137520, and a combination of MenB vaccine antigen genes, BAST-2988, have also been described.

The second leading clonal complex of the study isolates of N. meningitidis Y was cc92 (Fig 1). It is represented by three invasive and four carriage isolates and is quite heterogeneous in the phylogenetic network. One lineage of clonal complex cc92 is formed by two almost identical carriage isolates, 323/93 and 338/93, along with a more distant carriage isolate 59/93 (ST-92). Another lineage of cc92, groups two genetically closely related invasive isolates 200/94 and 449/95. A more distant relatedness to this lineage was evidenced for a recent invasive isolate, 37/18. The list of isolates of clonal complex cc92 ends with an outlier carriage isolate, 224/93 which is the only one to be assigned to a different sequence type, ST-94.

Six highly related invasive isolates form lineage cc167 in the phylogenetic network, which shows relatedness to lineage cc92. All cc167 isolates carry a single sequence type, ST-168, and BAST-384. In isolate 19/17, a single nucleotide deletion occurred in the variable region (VR) of the fetA gene, resulting in a reading frame shift and internal stop codon emergence. This mutated fetA allele, unable to produce functional peptide, was registered in the PubMLST database as allele 2816. Another comparatively heterogeneous lineage groups four ST-130 isolates. For isolate 105/07, a new allele variant of the fHbp gene (1079) was described, which differs from the original allele 302 by two synonymous substitutions, thus encoding the same peptide variant, 245. An outlier invasive isolate of clonal complex cc103 appears to be distantly related to the ST-130 lineage of isolates. Based on this outlier, a new sequence type, ST-11017, a new rST-169886, and a new combination of MenB vaccine antigen genes, BAST-2972, were defined. Even more distant relatedness to ST-130 isolates is seen in a lineage of three ST-3015 (ccUA) isolates recovered between 2003 and 2007. The last isolate from our collection– 61/18 (P1.21,16: F3-7; ST-1466 (cc174); rST-80732; BAST-14) is a phylogenetic outlier and does not show any relatedness to other isolates of N. meningitidis Y.

Czech and European isolates of N. meningitidis Y

The phylogenetic network (Fig 3A and 3B) shows the levels of relatedness among European invasive isolates of N. meningitidis Y, including Czech isolates. These were isolates from the following countries: UK (n = 612), Sweden (n = 112), France (n = 62), Germany (n = 49), the Netherlands (n = 46), Czech Republic (n = 35), Ireland (n = 29), Austria (n = 12), Norway (n = 11), Italy (n = 10), Finland (n = 6), Slovenia (n = 6), Malta (n = 3), Cyprus (n = 1), and Greece (n = 1).

Fig 3

Genetic diversity of N. meningitidis Y invasive isolates from the Czech Republic and European countries.

Isolates (n = 995) were collected from 1986 to 2020. Invasive isolates from Czech Republic (n = 35) are marked with a dark blue square and labelled by their NRL number, ST, and cc. A) Isolates are coloured according to detection year: 1986–1999 (n = 8), 2000–2009 (n = 28), 2010–2015 (n = 524), 2016–2020 (n = 435). B) Isolates are coloured according to the country of origin. The individual lineages of the cc23 clonal complex are marked by Roman numerals (I—IX).

The general view shows that one part of the European isolates of N. meningitidis Y form a large subpopulation of several lineages of clonal complex cc23 in the phylogenetic network, while the other part form separate genetic lineages consistent with the respective clonal complexes.

The large cc23 subpopulation consisting of several more or less related lineages includes 18 invasive isolates of clonal complex cc23 from the Czech Republic. All cc23 lineages are represented by isolates from all time periods, and none of them can be considered as historical or modern (Fig 3A). The dominance of isolates from the years 2010–2020 is caused by the overall low proportion of isolates from the years 1986–2009 in the phylogenetic analysis. An identical phylogenetic network that shows geographical origin of isolates in colour provides a completely different view (Fig 3B). A distant lineage (I), grouping mostly ST-23 isolates from the UK can be noticed. In three similar and genetically closely related lineages (II, III, and IV), the UK isolates are dominant again; however, isolates from neighbouring countries are also significantly represented. Most isolates from these three lineages belong to sequence type ST-1655. Another cc23 lineage comprises mostly Swedish ST-23 isolates (V). Close to the Swedish lineage is a geographically diverse lineage (VI) of isolates from continental Europe where ST-23 and sequence types derived from it are dominant. Apart from a cluster of six Czech isolates (251/01, 94/07, 121/08, 49/09, 4/11, and 32/13), this lineage includes primarily isolates from Germany, Austria, the Netherlands, and Slovenia. Czech isolates 63/10 and 42/13, which do not cluster together, belong to a genetically more distant lineage comprising mostly ST-23 isolates from France (VII). The last Czech cc23 isolate is 55/02 (ST-23), which, like the previous Czech phylogenetic network, is an outlier, and from its position it is evident that it is highly genetically distant from all other European cc23 isolates.

European isolates of N. meningitidis Y not belonging to cc23 show no significant trend in temporal distribution. The phylogenetic network which defines these isolates as genetically highly distant from cc23 subpopulation splits into three major lineages. The first one is a compact lineage, cc174, consisting exclusively of ST-1466 isolates including isolate 61/18 from the Czech Republic. The second lineage is highly heterogeneous, both geographically and genetically. It groups isolates ccUA, cc22, cc103, cc11, and cc865. The third major lineage in terms of heterogeneity appears in between the two former lineages. At the bottom, a sublineage of five cc92 isolates separates from it, formed by a cluster of three Czech isolates ST-92 along with a UK and Dutch isolates (ST-784). Other members of this third major lineage are almost exclusively cc167 isolates, including the cluster of six ST-168 isolates from the Czech Republic.

Czech and non-European isolates of N. meningitidis Y

The last phylogenetic network (Fig 4A and 4B) compares invasive isolates of N. meningitidis Y from the Czech Republic and non-European countries. In this selection, the MenY isolates from the USA are dominant (n = 315), followed by Japan (n = 24), New Zealand (n = 13), Mexico (n = 8), Canada (n = 4), Turkey (n = 3), China (n = 2), Morocco (n = 2), Burkina Faso (n = 2), Chile (n = 1), and Bangladesh (n = 1). Along with 35 isolates from the Czech Republic, the study set comprises 410 invasive isolates.

Fig 4

Genetic diversity of N. meningitidis Y invasive isolates from the Czech Republic and non-European countries.

Isolates (n = 410) were collected from 1993 to 2020. Invasive isolates from Czech Republic (n = 35) are marked with a dark blue square and labelled by their NRL number, ST, and cc. A) Isolates are coloured according to detection year: 1993–1999 (n = 14), 2000–2009 (n = 53), 2010–2015 (n = 213), 2016–2020 (n = 130). B) Isolates are coloured according to the country of origin. The individual lineages of the cc23 clonal complex are marked by Roman numerals (I–IV).

A general view of the phylogenetic network shows again a large subpopulation of cc23 isolates while isolates of N. meningitidis Y of other clonal complexes form several separate more or less numerous lineages on the opposite side.

Unlike the European cc23 isolates, non-European cc23 isolates appear less homogeneous. They form four separate, genetically distinct lineages. Furthermore, they differ in temporal distribution of isolates in individual lineages (Fig 4A). The first cc23 lineage (I) comprising exclusively of isolates from the USA and Mexico is represented mostly by isolates ST-3587 recovered after 2016 (Fig 4B). Another cc23 lineage (II) groups two main sister sublineages of isolates from the USA, with ST-23 and related ST-3532 being dominant. In addition to the two main sublineages, three other sublineages separate from the lineage II of cc23 isolates. One of these sublineages groups two clusters of 9 isolates from the Czech Republic (ST-569 and ST-1625) along with three isolates from the USA. Unlike the first major cc23 lineage, the second one is evenly represented by isolates from all time periods. The third cc23 lineage is geographically more diverse (III) and mostly assigned to sequence type ST-23. Two sublineages diverge from this lineage: one with dominant ST-23 groups isolates from the USA along with two Czech isolates and sublineage ST-1655 comprising isolates from the USA, Japan, New Zealand, and China. The remaining six Czech ST-23 isolates cluster together within the major lineage. The last, fourth cc23 lineage (IV) only includes two isolates, 55/02 (ST-23) from the Czech Republic collected in 2002, which so far occupied a completely separate position in the phylogenetic networks, along with a related American isolate, M28856 (ST-6519) from 2014.

Non-European isolates of N. meningitidis Y which do not belong to clonal complex cc23 again form several lineages genetically distant from each other on the phylogenetic network. In comparison with the European isolates, non-European isolates are less represented by other clonal complexes, as e.g., two genetic lineages are only formed by isolates from the Czech Republic (ST-92 and ST-130). The only exception is the richly represented lineage cc167, consisting mainly of American isolates ST-1624. Another lineage is represented exclusively by ST-1466 (cc174) isolates, including isolate 61/18 from the Czech Republic.

Discussion

We analysed WGS data of all N. meningitidis Y isolates received from IMD in the Czech Republic in the studied period. WGS showed a higher discrimination power and provided more accurate data on molecular characteristics and genetic relationships among invasive N. meningitidis isolates [18]. However, it is costly for the NRL to perform genomic surveillance for all N. meningitidis isolated from IMD and therefore we focus on serogroups that cause a high case fatality rate of this disease. Our study was focused on the analysis of WGS data of Czech isolates of N. meningitidis serogroup Y and their comparison with foreign data because this serogroup causes high IMD mortality worldwide and its increase has recently been recorded [6-14]. Comparison of WGS data of Czech isolates of N. meningitidis Y with foreign ones was also performed because in a previous study we found that the population of Czech isolates of N. meningitidis serogroup W was genetically different from European ones [17].

A limitation of our study is a low number of N. meningitidis Y isolates. However, this corresponds to the real epidemiological situation in the Czech Republic, i.e. low frequency of IMD caused by serogroup Y, although its rise has been recorded recently.

The European invasive isolates of N. meningitidis Y, including Czech isolates, can be considered as a relatively heterogeneous population. Although about two thirds of which are genetically closely related and belong to cc23, the remaining isolates are assigned to many other clonal complexes and show a very low relatedness to cc23 isolates and to each other. Isolates of N. meningitidis Y belonging to cc23 and other clonal complexes form sublineages within genetic lineages, defined mainly geographically. The population of invasive isolates of N. meningitidis Y appears to be constant over time compared to non-European isolates. The Czech isolates of N. meningitidis Y follow the trend observed for European isolates. They belong to several different clonal complexes and mostly cluster together within individual genetic lineages.

The non-European population of isolates of N. meningitidis Y appears to be less diverse in terms of clonal complexes as compared to the European one. Most isolates belong to complexes cc23 and cc167. A higher genetic diversity can be seen within individual clonal complexes–particularly in subpopulation cc23. The non-European population of N. meningitidis Y also shows a different time distribution of isolates. Within clonal complexes, several modern sublineages appear, containing mostly isolates collected after 2016. The Czech isolates of N. meningitidis Y, added to the non-European isolates for comparison, do not follow this non-European trend. Their clonal heterogeneity considerably contributes to the overall heterogeneity of the phylogenetic network, which contains Czech and non-European isolates.

The worldwide increase in IMD caused by serogroup Y has led to increased efforts to study N. meningitidis Y by the latest molecular methods. The analysis of WGS data on Swedish IMD isolates of N. meningitidis Y from 1995–2012 revealed two major branches of clonal complex cc23, one of which corresponded to YI strain type (ST-23) and the other to YII and YIII strain types [14]. The comparison of the Swedish WGS data with those from England, Wales, and Northern Ireland confirmed the spread of these two branches across continents over the last few years, although these branches were referred to differently in studies. IMD isolates of serogroup Y collected in England and Wales in 2007–2009 belonged to four major clonal complexes: cc23 (56%), cc174 (21%), cc167 (11%), and cc22 (8%). The rise in IMD caused by serogroup Y in 2009 was mainly due to ST-1655 (cc23) [10]. The variability of N. meningitidis Y cc23 was also reported for Italian IMD isolates from 2007–2013, assigned to nine different STs (ST-23, ST-10098, ST-1665, ST-10348, ST-9326, ST-2533, ST-9253, ST-3171, and ST-2692), with a predominance of ST-23 [13]. In our study, ST-23 was also prevalent among isolates of N. meningitidis Y cc23, but other STs (ST-1625, ST-569, and ST-8526) differed from those identified in the Italian study. Isolates ST-1655 (cc23) that caused the rise in IMD in England and Wales were not observed in our collection. IMD isolates of serogroup Y collected in Canada in 1999–2003 were assigned to two major clonal complexes: cc23 and cc167 [37]. The prevalence of clonal complexes cc23 and cc167 found in our study is consistent with the Canadian data, as is the prevalence of ST-23 in clonal complex cc23. The comparison of IMD isolates of serogroup Y from the United States, South Africa, and Israel collected in 1999–2002 showed the prevalence of two clonal complexes, cc23 and cc175. However, differences were found in the representation of clonal complexes between countries: the USA and Israel reported the prevalence of cc23, while in South Africa, the prevalence of cc175 was observed [38]. There was not a single cc175 isolate in our collection.

In conclusion, the WGS analysis showed the population of Czech N. meningitidis Y isolates as relatively heterogeneous, containing a large number of genetic lineages. The most represented clonal complex in Czech N. meningitidis Y isolates was cc23, which forms a separate lineage, distinct from all other isolates of N. meningitidis Y. Isolates of other clonal complexes show a very low relatedness to cc23 isolates and to each other. The comparison with foreign WGS data showed that within the main genetic lineages, which are defined by clonal complexes, Czech isolates of N. meningitidis Y, similar to European ones, mostly cluster together and form geographical sublineages. The Czech isolates of N. meningitidis Y generally follow the trend observed for European isolates in contrast to Czech isolates of serogroup W, for which a different trend was observed in our previous publication [17]. This result was one of the bases for updating the recommended vaccination strategy and in the Czech Republic the conjugated tetravaccine ACYW started to be used instead of the previous conjugated monovaccine C. In the future, we would like to prepare similar population studies with isolates of serogroups B and C, which are constantly dominant in the Czech Republic.

Serogroups of N. meningitidis causing invasive meningococcal disease, Czech Republic, 1993–2018, surveillance data.

(PDF)

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Clonal complexes of N. meningitidis Y causing invasive meningococcal disease, Czech Republic, 1993–2018, age groups, surveillance data.

(PDF)

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Clonal complexes of N. meningitidis Y causing invasive meningococcal disease, Czech Republic, 1993–2018, surveillance data.

(PDF)

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29 Oct 2021

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Reviewer #1: 1- line 33 -37 please rephrase it so it will be more clear

2-In the introduction we have to highlight the importance of our research why did you do sequencing for serogroup Y and not the main groups ,if there were increase in serogroup Y after introducing the MenC and MenB ,now as you mentioned there are 2 quadrivalent vaccines covering the Y serogroup,what is the outcome after doing WGS and the relation to the current vaccine.3-please try to summarize the description of each figure without going for much details. 4-in the discussion we have to mention our results and compare it with different countries

Reviewer #2: The manuscript entitled “Whole Genome Sequencing of Neisseria meningitidis Y isolates collected in the Czech Republic in 1993-2018” compares meningococcal serogroup Y genomes from the Czech Republic with other European and non-European genomes in order to relate the lineages in the Czech Republic to a global setting. Vaccination against serogroups ACWY and B is recommended in the Czech Republic and the study also includes characterization on theoretical vaccine coverage. Although the results from this study is an important contribution to the long-term epidemiological surveillance of meningococcal isolates in general, and the emergent serogroup Y in particular, the manuscript needs to be clarified and restructured and especially the results and discussion need a major overview according to the points suggested below.

Major points:

1. The aim of the study is not entirely clear to me and some analyses in this study are incompletely incorporated into the narrative. For example, the theoretical vaccine coverage BAST and MenDeVAR index is not described in M&M and it is not discussed in the discussion section what this information adds. Additionally, although very important, without a clearer aim it is unclear why the Czech isolates were compared to European isolates and then non-European isolates.

2. The results section lacks a clear narrative and is difficult to get through. Although there is no limit on word count, the manuscript is 6000 words long and seems to be unbalanced, 3 pages of introduction, 13 pages of results, 3 pages of discussion. The results section would benefit from being shortened and some parts moved to the discussion.

3. Figure S1 gives the reader an important sense of how the serogroup Y incidence, although increasing, is still very low in the Czech Republic compared to serogroups B and C. The authors could argue that it is rather the high mortality rate of serogroup Y that is the issue but this needs to be clearer as the introduction focuses on the increased serogroup Y IMD incidence.

4. Everything is relative but calling the serogroup Y isolates heterogeneous when 2/3 belonged to cc23 and then looking at non-European isolates which were even more heterogeneous, maybe then one would say that the Czech isolates were quite homogeneous? This could be re-phrased to be a more “soft” statement throughout the manuscript.

5. The discussion is incomplete and mostly mentions other studies. The most important findings should be highlighted and how the results adds to the field of research, possible limitations with the study and future directions should be added.

6. The manuscript would benefit from a general language revision, especially in regard to grammatical tense.

Minor points:

All figures would benefit from having the N= written in the figure legends instead of in the main text. Abbreviations NRL, ST and cc in the legends should be explained.

Line 20-22: The material and methods section of the abstract is somewhat short, it does not for example mention that the Czech isolates were compared to other countries.

Line 59: The cause of shifting meningococcal serogroup incidences in meningococci is not completely known and vaccination programmes is probably only part of the explanation, the sentence could be rephrased to e.g. “…partly due to vaccination programmes”.

Line 73: Please add a reference to this statement.

Line 78: The study was from 2015, not 2014.

Line 80: WGS was done in reference number 14.

Line 88: What is the timeline for the numbers on serogroup distribution, since 1993?

Introduction: please add numbers on IMD incidence in the Czech Republic. These would be interesting to see in conjunction to the vaccine strategy.

Line 128: There is no information about the healthy carriers, in which setting were these sampled? What was the purpose of these samples in the study?

Material and methods: The BAST, MenDeVAR index and rMLST are not mentioned.

Table 1: The entire table is not visible. The abbreviations “CAR” and “PAC” are not explained.

Line 215-216: Please give an estimation on how many the minimal genetic differences are.

Line 295-303: This should be mentioned in the Material and Methods section.

Line 479: Please use “n=3” instead of “3x ST-2880”, should be changed throughout the manuscript.

Line 494-503: This type of reasoning is better fitted for the discussion section rather than the results.

Line 515: The discussion starts with comparisons of which STs dominates in different countries, suggestion to add that the YI strain in Sweden belongs to ST-23.

Discussion: In the introduction it is stated that molecular characterization of serogroup Y provides background data for vaccination strategy, how the results from the current study will impact vaccination strategies could be discussed.

Fig 3-4: The roman letters are not explained in the figure legend.

Reviewer #3: Dear author,

Your work represents a nice work and adds valuable information of the epidemiologically situation based on whole genome sequencing of Neisseria meningitides serogroup Y isolates, collected in the Czech Republic during 1993-2018, and also its relation to other international isolates. The manuscript though needs extensive revisions, especially regarding the structure and focus of the scope of aim, i.e all parts regarding vaccination need to be shortened or in some places even taken away.

You can see more in detail below.

1. A specified aim is missing both in the manuscript and in the abstract.

2. The introduction is too long and need to be both structured and written in a logic way. For example, the section about vaccination is to detailed and needs to be shortened substantially, it should also be mentioned that the incidence and variation of causative serogroups varies over time not only due to vaccination but also natural fluctuations.

3. In M&M the collection of carrier isolates need to be described how it was done.

4. All subheadings (in the entire manuscript) need to be optimised and shortened, i.e “DNA extraction, WGS protocol, and WGS data analysis and visualization” could be Whole genome sequencing and analysis or even divided into sections.

5. The technically sound of the sequencing must be improved.

6. All abbreviations in the Tables and figures need to be explained in footnotes or used without abbreviation and print full name, especially in the headings (should be possible to read stand-alone).

7. The result section is too long and should only include the actual results and not new background information or interpretations. Do not repeat all results that can be found in tables and figures, just the most important findings.

8. The interpretations can be moved to the section of discussion. Also do not include all results from every country in the discussion only the comparison.

9. The conclusion (both in manuscript and abstract) needs to answer an aim (that first has to be defined…).

10. All figures need better solution.

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Reviewer #1: Yes: Manal Hamed

Reviewer #2: No

Reviewer #3: No

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7 Dec 2021

PONE-D-21-29616

Whole Genome Sequencing of Neisseria meningitidis Y isolates collected in the Czech Republic in 1993-2018

PLOS ONE

The authors thank the academic editor and three reviewers for their valuable comments and recommendations. Most of them were accepted - see the answers to the points below.

Response to the academic editor:

While your paper addresses an interesting question and provide valuable information of the epidemiologically situation of Neisseria meningitides, there are significant concern both about the presentation as well as the readability of the manuscript. In particular, the rationale of the study needs to be strengthen and the aim of the study need to be explained in the abstract and introduction. The writing was inadequate and accordingly, the data were not following a clear logical line of thought. In addition there were numerous issues identified where additional experimentation and documentation is needed, please see reviewers’ insightful comments below. On a more personal level, I also have a few issues with the manuscript that needs to be addressed, please see specific comments below. Furthermore, the quality of the language needs to be improved. Please have a fluent, preferably native, English-language speaker thoroughly copyedit your manuscript for language usage, spelling, and grammar. If you do not know anyone who can do this, we suggest you use a professional language editing or copyediting service.

These recommendations were mostly accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

Specific comments:

1. Line 49 – 56, combine these two paragraphs into one.

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

2 Line 158, please specify which ribosomal gene?

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

3. Expand the explanation of Table 1 further. For examples, Most prevalent is cc23 with 21 isolates (ST-23, 569,1625, and 8526). Followed by cc92 (ST-92 and ST-94) with 7 isolates. and cc167 (ST-168) with 6 isolates, as well as one isolate each for cc103 (ST11017) and cc174 (ST1466), etc.

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

4. Table 1 needs substantial editing to provide more relevant information. A few suggestion here:

1) Year of isolation is indicated in the strain ID? Is it necessary to have separate year of isolation?

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“. 2) Symptoms, you can probably delete this column and put * on the strains ID isolated from carrier and explained in the note; This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“. 3) Delete columns of serogroup and genogroup since all the isolates belong to the same serogroup and genogroup; This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

4) Add column to indicate clonal complex, BAST, and rST The columns indicating clonal complex, BAST and rST were already in Table 1 that was in the first version of the submitted manuscript. Unfortunately, they were not visible. In the revised version of the manuscript which we are now submitting, Table 1 was divided into Table 1 and Table 2 and all columns are visible.

5. Note for Table 1 needs significant editing to provide relevant information: 1) Unless the authors added column indicating cc, the explanation for cc and UA is unnecessary; As explained above, unfortunately not all Table 1 columns were visible in the first version of the manuscript. In the revised version of the manuscript which we are now submitting, Table 1 was divided into Table 1 and Table 2 and all columns are visible. The explanation for cc and UA is necessary for Table 1. 2) Line 183 – 185, not sure that these are relevant to the current Table 1. As explained above, unfortunately not all Table 1 columns were visible in the first version of the manuscript. In the revised version of the manuscript which we are now submitting, Table 1 was divided into Table 1 and Table 2 and all columns are visible. This text is relevant for Table 1 and Table 2.

6. Line 187 – 193, combine these two paragraphs into one.

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

7. Line 307 – 310, are these sentence referring to one of the figures?

As explained above, unfortunately not all Table 1 columns were visible in the first version of the manuscript. In the revised version of the manuscript which we are now submitting, Table 1 was divided into Table 1 and Table 2 and all columns are visible. This text is relevant for Table 2.

8. Line 314, please explain Trumenba.

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

9. Line 317 – 322, These are confusing sentences. Not sure what the authors wish to convey?

As explained above, unfortunately not all Table 1 columns were visible in the first version of the manuscript. In the revised version of the manuscript which we are now submitting, Table 1 was divided into Table 1 and Table 2 and all columns are visible. This text is relevant for Table 2.

10. Line 356 – 357, awkward sentence, please rephrase for clarity.

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

11. Discussion needs to focus on discussing the results first and how it compared to other literatures.

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

Response to the Reviewer #1:

1- line 33 -37 please rephrase it so it will be more clear

2-In the introduction we have to highlight the importance of our research why did you do sequencing for serogroup Y and not the main groups ,if there were increase in serogroup Y after introducing the MenC and MenB ,now as you mentioned there are 2 quadrivalent vaccines covering the Y serogroup,what is the outcome after doing WGS and the relation to the current vaccine. 3-please try to summarize the description of each figure without going for much details. 4-in the discussion we have to mention our results and compare it with different countries

These recommendations were accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

Response to the Reviewer #2:

The manuscript entitled “Whole Genome Sequencing of Neisseria meningitidis Y isolates collected in the Czech Republic in 1993-2018” compares meningococcal serogroup Y genomes from the Czech Republic with other European and non-European genomes in order to relate the lineages in the Czech Republic to a global setting. Vaccination against serogroups ACWY and B is recommended in the Czech Republic and the study also includes characterization on theoretical vaccine coverage. Although the results from this study is an important contribution to the long-term epidemiological surveillance of meningococcal isolates in general, and the emergent serogroup Y in particular, the manuscript needs to be clarified and restructured and especially the results and discussion need a major overview according to the points suggested below.

These recommendations were accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

Major points:

1. The aim of the study is not entirely clear to me and some analyses in this study are incompletely incorporated into the narrative. For example, the theoretical vaccine coverage BAST and MenDeVAR index is not described in M&M and it is not discussed in the discussion section what this information adds. Additionally, although very important, without a clearer aim it is unclear why the Czech isolates were compared to European isolates and then non-European isolates.

These recommendations were accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

2. The results section lacks a clear narrative and is difficult to get through. Although there is no limit on word count, the manuscript is 6000 words long and seems to be unbalanced, 3 pages of introduction, 13 pages of results, 3 pages of discussion. The results section would benefit from being shortened and some parts moved to the discussion.

These recommendations were accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

3. Figure S1 gives the reader an important sense of how the serogroup Y incidence, although increasing, is still very low in the Czech Republic compared to serogroups B and C. The authors could argue that it is rather the high mortality rate of serogroup Y that is the issue but this needs to be clearer as the introduction focuses on the increased serogroup Y IMD incidence.

These recommendations were accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

4. Everything is relative but calling the serogroup Y isolates heterogeneous when 2/3 belonged to cc23 and then looking at non-European isolates which were even more heterogeneous, maybe then one would say that the Czech isolates were quite homogeneous? This could be re-phrased to be a more “soft” statement throughout the manuscript.

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

5. The discussion is incomplete and mostly mentions other studies. The most important findings should be highlighted and how the results adds to the field of research, possible limitations with the study and future directions should be added.

These recommendations were accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

6. The manuscript would benefit from a general language revision, especially in regard to grammatical tense.

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

Minor points:

All figures would benefit from having the N= written in the figure legends instead of in the main text.

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

Abbreviations NRL, ST and cc in the legends should be explained.

Abbreviations NRL, ST and cc is explained in Materials and Methods – please see in the enclosure „Revised Manuscript with Track Changes“.

Line 20-22: The material and methods section of the abstract is somewhat short, it does not for example mention that the Czech isolates were compared to other countries.

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

Line 59: The cause of shifting meningococcal serogroup incidences in meningococci is not completely known and vaccination programmes is probably only part of the explanation, the sentence could be rephrased to e.g. “…partly due to vaccination programmes”.

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

Line 73: Please add a reference to this statement.

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

Line 78: The study was from 2015, not 2014.

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

Line 80: WGS was done in reference number 14.

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

Line 88: What is the timeline for the numbers on serogroup distribution, since 1993?

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

Introduction: please add numbers on IMD incidence in the Czech Republic. These would be interesting to see in conjunction to the vaccine strategy.

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

Line 128: There is no information about the healthy carriers, in which setting were these sampled? What was the purpose of these samples in the study?

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

Material and methods: The BAST, MenDeVAR index and rMLST are not mentioned.

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

Table 1: The entire table is not visible. The abbreviations “CAR” and “PAC” are not explained.

As explained above, unfortunately not all Table 1 columns were visible in the first version of the manuscript. In the revised version of the manuscript which we are now submitting, Table 1 was divided into Table 1 and Table 2 and all columns are visible. The column with the abbreviations CAR and PAC was deleted according to the recommendation of the academic editor. Isolates from IMD versus isolates from carriers are distinguished by bold in the column Strain IDs in Table 1 and Table 2.

Line 215-216: Please give an estimation on how many the minimal genetic differences are.

This comment was accepted, the part of sentence about minimal genetic differences was deleted – please see in the enclosure „Revised Manuscript with Track Changes“.

Line 295-303: This should be mentioned in the Material and Methods section.

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

Line 479: Please use “n=3” instead of “3x ST-2880”, should be changed throughout the manuscript.

The expressions such as "3x" have been deleted from the text throughout the manuscript as part of the recommended shortening of the text.

Line 494-503: This type of reasoning is better fitted for the discussion section rather than the results.

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

Line 515: The discussion starts with comparisons of which STs dominates in different countries, suggestion to add that the YI strain in Sweden belongs to ST-23.

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

Discussion: In the introduction it is stated that molecular characterization of serogroup Y provides background data for vaccination strategy, how the results from the current study will impact vaccination strategies could be discussed.

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

Fig 3-4: The roman letters are not explained in the figure legend.

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

Response to the Reviewer #3:

Your work represents a nice work and adds valuable information of the epidemiologically situation based on whole genome sequencing of Neisseria meningitides serogroup Y isolates, collected in the Czech Republic during 1993-2018, and also its relation to other international isolates. The manuscript though needs extensive revisions, especially regarding the structure and focus of the scope of aim, i.e all parts regarding vaccination need to be shortened or in some places even taken away.

These recommendations were accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

You can see more in detail below.

1. A specified aim is missing both in the manuscript and in the abstract.

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

2. The introduction is too long and need to be both structured and written in a logic way. For example, the section about vaccination is to detailed and needs to be shortened substantially, it should also be mentioned that the incidence and variation of causative serogroups varies over time not only due to vaccination but also natural fluctuations.

These recommendations were accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

3. In M&M the collection of carrier isolates need to be described how it was done.

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

4. All subheadings (in the entire manuscript) need to be optimised and shortened, i.e “DNA extraction, WGS protocol, and WGS data analysis and visualization” could be Whole genome sequencing and analysis or even divided into sections.

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

5. The technically sound of the sequencing must be improved.

We do not understand this recommendation. The sequencing used in the present study have been described in detail previously, as is mentioned in M&M part [ref. 17 and 18].

6. All abbreviations in the Tables and figures need to be explained in footnotes or used without abbreviation and print full name, especially in the headings (should be possible to read stand-alone).

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

7. The result section is too long and should only include the actual results and not new background information or interpretations. Do not repeat all results that can be found in tables and figures, just the most important findings.

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

8. The interpretations can be moved to the section of discussion. Also do not include all results from every country in the discussion only the comparison.

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

9. The conclusion (both in manuscript and abstract) needs to answer an aim (that first has to be defined…).

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

10. All figures need better solution.

When submitting the corrected version of the manuscript, we have uploaded our figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool https://pacev2.apexcovantage.com/ to be sure that our figures meet PLOS requirements.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

3 Jan 2022

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

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We look forward to receiving your revised manuscript.

Kind regards,

Baochuan Lin, Ph.D.

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: (No Response)

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: (No Response)

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: (No Response)

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: (No Response)

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Please see attached comments and recommendations

I want to thank the authors for the interesting research and for the meticulous detailed points mentioned in the manuscript.I have few points some are recommendations and others are inquiries .

This manuscript has been re-written and reads much better as suggested by the previous reviewers . However further editing for clarity would be advised.

Abstract:

Line 38-39: The WGS has been described previously (This Phrase can be removed and mentioned in the M&M)

Any mention about studying the relatedness to vaccine strains in the abstract ?whether in the aim ,results ?

Introduction:

Line 105-109

The Swedish isolates comment can be removed from the introduction as you already mentioned it in the discussion

Methods

Paragraph from line 208 to 226 : would you clarify why you choose Vaccine covering group B ,you mentioned there are some protection but why we are studying vaccine covering group B and you have already vaccine covering Y strains why these vaccines were not studied ? would you elaborate more in this point .

Results:

Paragraph starting by line 269,these results of similarities between vaccines for Men B and the studied isolates, what is the interpretation of these results ,are you going to recommend these vaccines instead of the current vaccines for Y strains ?what is your recommendation after mentioning the color coding similarities

Is it possible to mention this in the discussion part and how these results will update the vaccine strategy .

Line 347: would you mention at the beginning how many invasive and how many carriers having the CC92 and the same for CC23

Too much details for the infrequent CC ,better to summarize this part only mentioning the main findings.

Fig3A and 3B have the same title ?

Paragraph started by line 495: Too much details ,you can summarize this paragraph as this is the infrequent non CC23,you can put all non 23CC in one paragraph.

Paragraph started by line 567: summarize lineage ii the main findings you cannot describe each and every sub-lineage ,only general description because everything is shown in the figure so you mention what you cannot describe in the figure

Discussion :

Line 648 : I think this was mentioned before by one of the reviewer that if 2/3 of isolates are CC23 how can we considered that as heterogeneous?

Line657: The non-European population of isolates of N. meningitidis Y appears to be less diverse or more diverse

Line 670 to 679 would you rephrase the sentence please, the meaning is not clear (the whole paragraph need to be rewritten )

Line 744 to 748: how these results will update on the strategy of vaccine coverage in Czech Republic.

Reviewer #2: The authors have made substantial changes to the manuscript and many of my points have been addressed, however, some new points have arisen:

(the lines refer to the version with tracked changes)

Lines 218-223, it is a bit confusing listing the colors according to MenDeVar index here without a reference to a table. The colors could be left out in the material and method section and clarified in the table instead.

Line 723-724, it is unclear why cc175 is mentioned in this paragraph, is this not related to the previous paragraph and should be moved to line 717?

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: Yes: Manal Mahmoud Hamed

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Submitted filename: Recommendations for manuscript.docx

Click here for additional data file.

20 Jan 2022

PONE-D-21-29616R1

Whole genome sequencing of Neisseria meningitidis Y isolates collected in the Czech Republic in 1993-2018

PLOS ONE

The authors thank the academic editor and two reviewers for their valuable comments and recommendations. Most of them were accepted - see the answers to the points below.

Response to the academic editor:

Both reviewers agreed that the revised manuscript showed substantial improvement, however, some new points have arisen and need to be addressed carefully. Please see reviewers’ insightful comments below. Also, there are numerous issues where additional clarification is needed (specific comments).

These recommendations were mostly accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

In addition, the quality of the language still needs to be improved. We suggest you thoroughly copyedit your manuscript for language usage, spelling, and grammar. If you do not know anyone who can help you do this, you may wish to consider employing a professional scientific editing service.

The English proofing of the second revised version of the manuscript was carried out by Keith Jolley of the University of Oxford, who, in addition to being a native speaker, is also a world-renowned expert in whole genome sequencing of Neisseria meningitidis.

Specific comments:

1. Line 193 – 195: The statement “Analysis of the theoretical…” is not relevant to the manuscript, suggest deletion.

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

2. Line 215: Suggest adding the following statement "The highest incidence of IMD caused by N. meningitidis Y in Czech Republic is in adolescents/young adults (age 15 - 19), followed by older adults (≥65 years of age) and in younger children (1 - 4 years old). (Fig. S3)” Suggest changing to Fig. S3 to S2 in order to reflect the fact that Fig. S3 appeared first.

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

3. Line 249: Suggest adding the following statement “The highest incidence of IMD caused by N. meningitidis Y in Czech Republic is in adolescents/young adults (age 15 - 19), followed by older adults (≥65 years of age) and in younger children (1 - 4 years old)”

This information would already be duplicated, given that we have already added the age information above, based on a previous recommendation.

4. Line 277: Are you referred to fig.2? If so, please mentioned here.

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

5. Line 277: Suggest changing “A more detailed representation shows…” to “A more detailed phylogenetic analysis of cc23 isolates (Fig. 2) showed…”

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

6. Line 334: It will also be nice to break out how many isolates are from 1986 - 1999, 2000 - 2009, 2010 - 2015, 2016 – 2020.

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

7. Line 336 – 343: Combine Fig. 3A and 3B legend into one. Fig 3. Genetic diversity of N. meningitidis Y invasive isolates from the Czech Republic and European countries. A) Genetic diversity of N. meningitidis Y isolates collected from 1986 to 2020: Isolates (n = 995) are coloured according to detection year. Invasive isolates from Czech Republic (n = 35) are marked with a dark blue square and labelled by their NRL number, ST, and cc. B) Genetic relationship of N. meningitidis Y isolates based on geographic origins: Isolates (n = 995) are coloured according to the country of origin. The individual lineages of the cc23 clonal complex are marked by Roman numerals (I – IX). In addition, suggest including similar coloring scheme for Fig. 3B.

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“. We do not understand the additional suggestion to include similar colors in Fig. 3B, because Fig. 3A presents different periods while Fig. 3B presents different countries.

8. Line 352 – 354: Fig. 3A seems to show that cc23 were more prevalent/diversified from 2010 - 2020. Only one from 1986 - 1999 and few from 2000 - 2010 which does not collaborate the statement here. Please clarify.

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

9. Line 354: Change “…all time period” to “…all time period (Fig. 3B)”. Also suggest using similar coloring scheme for Fig. 3B.

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“. We do not understand the additional suggestion to include similar colors in Fig. 3B, because Fig. 3A presents different periods while Fig. 3B presents different countries.

10. Line 402: Similarly, it will also be nice to break out how many isolates are from 1993 - 1999, 2000 - 2009, 2010 - 2015, 2016 – 2020.

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

11. Line 404 – 413: Similarly, please combine Fig. 4A and 4B legend into one. Fig 4. Genetic diversity of N. meningitidis Y invasive isolates from the Czech Republic and non-European countries. A) Genetic diversity of N. meningitidis Y isolates collected from 1993 to 2020: Isolates (n = 410) are coloured according to detection year. Invasive isolates from Czech Republic (n = 35) are marked with a dark blue square and labelled by their NRL number, ST, and cc. B) Genetic relationship of N. meningitidis Y isolates based on geographic origins: Isolates (n = 410) are coloured according to the country of origin. The individual lineages of the cc23 clonal complex are marked by Roman numerals (I – IV). Invasive isolates from Czech Republic (n =35) are marked with a dark blue square and labelled by their NRL number, ST, and cc. Also, Fig.4B should retain the same coloring as Fig. 4A as well.

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“. We do not understand the additional suggestion to include similar colors in Fig. 4B, because Fig. 4A presents different periods while Fig. 4B presents different countries.

12. Line 514: Why discuss the cc23 in Sweden? Does this correlate with Czech?

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

13. Discussion: Length discussion of cc23 in Sweden, England, Wales, USA, and Canada. Not sure what the authors wish to convey. Suggest consolidating these sections and shorten the discussion.

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

Response to the Reviewer #1:

Abstract:

Line 38-39: The WGS has been described previously (This Phrase can be removed and mentioned in the M&M)

Any mention about studying the relatedness to vaccine strains in the abstract ?whether in the aim ,results ?

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

Introduction:

Line 105-109

The Swedish isolates comment can be removed from the introduction as you already mentioned it in the discussion

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

Methods

Paragraph from line 208 to 226 : would you clarify why you choose Vaccine covering group B ,you mentioned there are some protection but why we are studying vaccine covering group B and you have already vaccine covering Y strains why these vaccines were not studied ? would you elaborate more in this point .

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

Results:

Paragraph starting by line 269,these results of similarities between vaccines for Men B and the studied isolates, what is the interpretation of these results ,are you going to recommend these vaccines instead of the current vaccines for Y strains ? what is your recommendation after mentioning the color coding similarities

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

Is it possible to mention this in the discussion part and how these results will update the vaccine strategy .

This was already stated in the previous version of the manuscript: …in the Czech Republic the conjugated tetravaccine ACYW started to be used instead of the previous conjugated monovaccine C. - so we leave it unchanged.

Line 347: would you mention at the beginning how many invasive and how many carriers having the CC92 and the same for CC23

This was already stated in the previous version for cc92 - so we are adding it also for cc23 – please see in the enclosure „Revised Manuscript with Track Changes“.

Too much details for the infrequent CC ,better to summarize this part only mentioning the main findings.

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

Fig3A and 3B have the same title ?

This recommendation is similar to the recommendation of the academic editor (see above) and was accepted: the title of Fig. 3A and 3B has been merged together – please see in the enclosure „Revised Manuscript with Track Changes“.

Paragraph started by line 495: Too much details ,you can summarize this paragraph as this is the infrequent non CC23,you can put all non 23CC in one paragraph.

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

Paragraph started by line 567: summarize lineage ii the main findings you cannot describe each and every sub-lineage ,only general description because everything is shown in the figure so you mention what you cannot describe in the figure

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

Discussion :

Line 648 : I think this was mentioned before by one of the reviewer that if 2/3 of isolates are CC23 how can we considered that as heterogeneous?

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

Line657: The non-European population of isolates of N. meningitidis Y appears to be less diverse or more diverse

It was already stated in the previous version of the manuscript that the non-European population appears to be less diverse - so we leave it unchanged.

Line 670 to 679 would you rephrase the sentence please, the meaning is not clear (the whole paragraph need to be rewritten )

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

Line 744 to 748: how these results will update on the strategy of vaccine coverage in Czech Republic.

This was already stated in the previous version of the manuscript: …in the Czech Republic the conjugated tetravaccine ACYW started to be used instead of the previous conjugated monovaccine C. - so we leave it unchanged.

Response to the Reviewer #2:

Lines 218-223, it is a bit confusing listing the colors according to MenDeVar index here without a reference to a table. The colors could be left out in the material and method section and clarified in the table instead.

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

Line 723-724, it is unclear why cc175 is mentioned in this paragraph, is this not related to the previous paragraph and should be moved to line 717?

This recommendation was accepted – please see in the enclosure „Revised Manuscript with Track Changes“.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

23 Feb 2022

Whole genome sequencing of Neisseria meningitidis Y isolates collected in the Czech Republic in 1993-2018

PONE-D-21-29616R2

Dear Dr. Krizova,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once you corrected the following errors and it meets all outstanding technical requirements. 1) Line 205 - 206, (ST-23, n = 7; ST-569, n = 4; ST-1625, n = 6; and ST-8526, n = 1). These numbers do not add up to 21 isolates, please correct.  2) Line 348, extra period at the end of the sentence, please correct.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Baochuan Lin, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: (No Response)

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: (No Response)

**********

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Reviewer #2: (No Response)

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Reviewer #2: No

2 Mar 2022

PONE-D-21-29616R2

Whole genome sequencing of Neisseria meningitidis Y isolates collected in the Czech Republic in 1993-2018

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