Genomic analysis of virulence factors and antimicrobial resistance of group B Streptococcus isolated from pregnant women in northeastern Mexico.

INTRODUCTION: Group B Streptococcus (GBS) causes infections in women during pregnancy and puerperium and invasive infections in newborns. The genes lmb, cylE, scpB, and hvgA are involved with increased virulence of GBS, and hypervirulent clones have been identified in different regions. In addition, increasing resistance of GBS to macrolides and lincosamides has been reported, so knowing the patterns of antibiotic resistance may be necessary to prevent and treat GBS infections. This study aimed to identify virulence genes and antibiotic resistance associated with GBS colonization in pregnant women from northeastern Mexico.
METHODS: Pregnant women with 35-37 weeks of gestation underwent recto-vaginal swabbing. One swab was inoculated into Todd-Hewitt broth supplemented with gentamicin and nalidixic acid, a second swab was inoculated into LIM enrichment broth, and a third swab was submerged into a transport medium. All samples were subcultured onto blood agar. After overnight incubation, suggestive colonies with or without hemolysis were analyzed to confirm GBS identification by Gram staining, catalase test, hippurate hydrolysis, CAMP test, and incubation in a chromogenic medium. We used latex agglutination to confirm and serotype GBS isolates. Antibiotic resistance patterns were assessed by Vitek 2 and disk diffusion. Periumbilical, rectal and nasopharyngeal swabs were collected from some newborns of colonized mothers. All colonized women and their newborns were followed up for three months to assess the development of disease attributable to GBS. Draft genomes of all GBS isolates were obtained by whole-genome sequencing. In addition, bioinformatic analysis to identify genes encoding capsular polysaccharides and virulence factors was performed using BRIG, while antibiotic resistance genes were identified using the CARD database.
RESULTS: We found 17 GBS colonized women out of 1154 pregnant women (1.47%). None of the six newborns sampled were colonized, and no complications due to GBS were detected in pregnant women or newborns. Three isolates were serotype I, 5 serotype II, 3 serotype III, 4 serotype IV, and 2 serotype V. Ten distinct virulence gene profiles were identified, being scpB, lmb, fbsA, acp, PI-1, PI-2a, cylE the most common (3/14, 21%). The virulence genes identified were scpB, lmb, cylE, PI-1, fbsA, PI-2a, acp, fbsB, PI-2b, and hvgA. We identified resistance to tetracycline in 65% (11/17) of the isolates, intermediate susceptibility to clindamycin in 41% (7/17), and reduced susceptibility to ampicillin in 23.5% (4/17). The tetM gene associated to tetracyclines resistance was found in 79% (11/14) and the mel and mefA genes associated to macrolides resistance in 7% (1/14).
CONCLUSIONS: The low prevalence of colonization and the non-occurrence of mother-to-child transmission suggest that the intentional search for GBS colonization in this population is not justified. Our results also suggest that risk factors should guide the use of intrapartum antibiotic prophylaxis. The detection of strains with genes coding virulence factors means that clones with pathogenic potential circulates in this region. On the other hand, the identification of decreased susceptibility to antibiotics from different antimicrobial categories shows the importance of adequately knowing the resistance patterns to prevent and to treat GBS perinatal infection.

Introduction

Group B Streptococcus (GBS; Streptococcus agalactiae) colonizes the human genitourinary and gastrointestinal tract and causes a variety of infectious processes in pregnant women, such as asymptomatic bacteriuria, urinary tract infection, bacteremia, pneumonia, meningitis, endocarditis, sepsis, and various obstetric complications as spontaneous abortion, premature labor, chorioamnionitis, endometritis, stillbirth, and neonatal and maternal death. In addition, according to the onset of clinical manifestations, GBS infection in newborns (NB) can present with bacteremia, pneumonia, and sepsis as early-onset disease, late-onset disease, or late-late-onset disease [1, 2]. Up to 40% of pregnant women present GBS colonization, and 1 to 2% of NB may develop infection by this microorganism [1, 3]. The main risk factor in developing GBS invasive neonatal disease is woman’s recto-vaginal colonization during childbirth [4].

GBS is considered an uncommon cause of perinatal infections in Mexico and other Latin American countries [5]. Unlike developed countries, in Mexico, neither the intentional search for GBS colonization in pregnant women nor the administration of intrapartum antibiotic prophylaxis (IAP) to prevent perinatal infection by this microorganism is an established practice [6]. However, a study in Mexico reported high exposure to GBS in reproductive age women (90%) [7]. GBS colonization of pregnant women is reported in up to 13% in Mexico and between 10 and 40% worldwide [1, 8].

Previous studies reported several factors making GBS more virulent and resistant to antibiotics. The most studied virulence factor is the capsular polysaccharide (Cps), which defines GBS serotypes (Ia, Ib, II-IX) and contributes to evade the immune system. However, other factors, such as laminin-binding protein (Lmb), fibrinogens (Fbs), hypervirulent adhesin (HvgA), and alpha-C protein (ACP), are associated with adherence and cell invasion [9-11]. In addition, an increasing GBS resistance to macrolides, lincosamides, and tetracyclines has been reported by several authors worldwide [12, 13]. Multidrug resistance has been increased all over the world that is considered a public health threat. Several recent investigations reported the emergence of multidrug-resistant bacterial pathogens from different origins, including humans, birds, cattle, and fish, that increase the need for routine application of antimicrobial susceptibility testing to detect the antibiotic of choice and the screening of the emerging MDR strains [13-17]. The aim of the present study was to explore the presence of virulence and antibiotic resistance genes in GBS associated with colonization in pregnant women in a population from northeastern Mexico.

Material and methods

Design, participants, and clinical sampling

A cross-sectional study and subsequently a longitudinal study were conducted from April 2017 to December 2018 including pregnant women with 35–37 weeks of gestation in prenatal care at the Hospital de Ginecología y Obstetricia No. 23 of the Instituto Mexicano del Seguro Social (IMSS). Written informed consent was obtained from all participants. The study was approved by the IMSS Scientific Research National Committee (R-2014-785-069). The sample collection was performed taking a recto-vaginal swab according to the recommendations of the Centers for Disease Control and Prevention (CDC) of the United States of America (USA). Sample collection procedures are available in protocols.io at dx.doi.org/10.17504/protocols.io.b4ytqxwn. In addition, we collected demographic data, including the socioeconomic status using the Graffar-Méndez scale, previously validated and used in the Mexican population [18]. We also register the number of pregnancies, gestational age, route of delivery, type of delivery (dystocic or eutocic), premature rupture of membranes, asphyxia at birth, and whether or not GBS colonized the NB.

Isolation and presumptive identification

One swab was inoculated into Todd-Hewitt broth supplemented with gentamicin 8 μg/ml and nalidixic acid 15 μg/ml (THB, Becton-Dickinson, NJ, USA). A second swab was inoculated into LIM enrichment broth (Todd-Hewitt broth with 1% yeast extract, 15 μg/ml nalidixic acid, and 10 μg/ml colistin, Becton-Dickinson, New Jersey, USA). Finally, we submerged the third swab into a transport medium (ESwab, Copan Diagnostics, Brescia, Italy). The first two swabs were incubated 18–24 hours at 37 °C in 5% CO2, then those 2 tubes along with the swab inside the transport medium were subcultured onto Petri dishes with Columbia agar (OXOID LTD, Basingstoke, Hampshire, England) with 5% sheep erythrocytes and incubated under the same conditions. After overnight incubation, suggestive colonies with or without hemolysis were analyzed to confirm GBS identification [19, 20]. GBS isolation procedure used is available in protocols.io at dx.doi.org/10.17504/protocols.io.b4ytqxwn.

GBS identification and serotyping

The criteria for GBS identification were a) Gram-positive coccus; b) negative catalase test; c) positive hippurate hydrolysis; d) CAMP test with wedge-shaped hemolysis in the presence of Staphylococcus aureus ATCC 25923; and e) growth with color change from white to orange pigment after 18–24 h incubation in the Strep B Carrot Broth® chromogenic medium (Hardy Diagnostics®, Santa María, CA, USA). Serotyping was performed by latex agglutination following the manufacturer’s instructions (ImmuLex® Strep-B Latex, Statens Serum Institute, Copenhagen, Denmark). Confirmed isolates were stored at -80 °C [19, 21]. GBS identification and serotyping procedures are available in protocols.io at dx.doi.org/10.17504/protocols.io.b4ytqxwn, dx.doi.org/10.17504/protocols.io.b4ygqxtw, dx.doi.org/10.17504/protocols.io.b4yjqxun, dx.doi.org/10.17504/protocols.io.b4ynqxve, dx.doi.org/10.17504/protocols.io.b4ymqxu6, and dx.doi.org/10.17504/protocols.io.b4ysqxwe.

Antimicrobial resistance profile of the recovered GBS isolates

The categories of antimicrobials tested were fluoroquinolones, glycopeptides, glycylcyclines, oxazolidinones, penicillins, tetracyclines, in addition to erythromycin and clindamycin [22]. Antibiotic susceptibility testing was performed in the VITEK 2 system (BioMérieux, Marcy-L’Etoile, France) with 1.5x107 CFU/ml of each GBS isolate loaded into the AST-GP75 card containing ampicillin, levofloxacin, moxifloxacin, linezolid, vancomycin, tetracycline, and tigecycline, according to the manufacturer’s instructions. In addition, susceptibility to erythromycin and clindamycin was evaluated by standardized agar diffusion tests using discs with these antibiotics (Titan Biotech, Bhiwadi, Alwar, Rajasthan). The results were assessed by measuring the diameter of the inhibition zone and interpreted according to the CLSI guideline [23].

Genomic DNA extraction and whole-genome sequencing

GBS isolates were cultured overnight at 37 °C in 5% CO2 in 3 ml of THB. Cellular pellets generated by centrifugation (6,000 g for 10 min at 10°C) were incubated with 180 μL of 50 mg/ml lysozyme for 60 min at 37 °C. The isolation of genomic DNA (gDNA) was made using the QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany). The concentration of gDNA was assessed using the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) on the Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA), and libraries were prepared with the Nextera DNA Flex Library Prep Kit (Illumina Inc. San Diego, CA, USA) following manufacturer’s instructions. Pooled DNA library was deposited on the sequencing cartridge (MiSeq Reagen Kit V2, Micro Flow Cell 300 cycles; Illumina Inc. San Diego, CA, USA) and sequenced in the MiSeq (Illumina Inc. San Diego, CA, USA) instrument.

Bioinformatic data processing

The quality of raw sequence data reads was validated using the 0.11.8 version of the FastQC program [24], and draft genomes were de novo assembled with the A5-miseq program [25]. The Quast program was used to evaluate quality details of the assemblies, such as contigs quantity and length [26]. Assemblies with poor quality parameters were subjected to a QC-Filtering pre-cleaning script. Genome annotation was made with the Prokka software [27], which identifies genes and their products by homology analysis in prokaryotic genomes. The final identification and mapping of the GBS genes coding for the capsular polysaccharide and the virulence factors FbsA, FbsB, Lmb, ACP, PI-1, PI-2a, PI-2b, HvgA, CylE, and ScpB were performed by blast homology analysis with BRIG software [28]. The identification of antibiotic resistance genes was performed by comparison using the CARD database [29].

Statistical analysis

The descriptive analysis of quantitative variables was carried out using medians with ranges and that of categorical variables with absolute frequencies and percentages. Differences between women colonized and not colonized by GBS in categorical variables were measured by either the X2 test or Fisher’s exact test and quantitative variables by the Mann-Whitney U test. The odds ratio and its 95% confidence interval were also measured. A p-value <0.05 was considered statistically significant. The analysis was made using the IBM SPSS Statistics software (Version 20; SPSS Inc., Armonk, NY, USA).

Ethics

The study was approved and supervised by the National Committee for Scientific Research (Registration number: R-2014-785-069) of the Instituto Mexicano del Seguro Social. All enrolled women provided written informed consent before inclusion in the study.

Results

Obstetric characteristics and GBS colonization

We first sought to determine the prevalence of GBS in the studied population, including 1,154 pregnant women. Seventeen women were colonized by GBS for a prevalence of 1.47%. Most of women were under 30 years of age (73%), 15 of the 17 colonized women belonged to this same age group (88%). Out of the total sample, 352 (30.9%) were primiparous; instead, 8 (47.1%) out of the 17 women colonized by GBS were also in their first pregnancy. We did not record the age of 16 non-colonized women and the number of pregnancies of 13 non-colonized women. Among the non-colonized women, 49 (4.3%) had obesity, 209 (18.4%) gestational diabetes, and 21 (1.8%) type 2 diabetes (Table 1).

Table 1

Sociodemographic and obstetric characteristics of 1154 pregnant women classified according to the status of group B Streptococcus (GBS) colonization.

		GBS Colonization
	Total (n = 1154)	Yes (n = 17)	No (n = 1137)	RM (IC95%)	p
Age (years)	0 27 (14–43)	25 (19–37)	0027 (14–43)	-	0.113
Age group
≤30 years	0833 (73.2%)	15 (88.2%)	0818 (73.0%)	2.77 (0.63–12.22)	0.125
>30 years	0305 (26.8%)	02 (11.8%)	0303 (27.0%)
Socioeconomic class*
Middle, middle/low, or low	0138 (12.0%)	04 (23.5%)	0134 (11.8%)	2.30 (0.74–7.16)	0.135
Other	1016 (88.0%)	13 (76.5%)	1003 (88.2%)
No. de pregnancies
1	0352 (30.9%)	008 (47.1%)	0344 (30.6%)	2.01 (0.77–5.26)	0.118
2 or more	0789 (69.1%)	009 (52.9%)	780 (69.4%)
Co-morbidities
Obesity	0051 (4.4%)	002 (11.8%)	0049 (4.3%)	2.96 (0.65–13.30)	0.171
Type 2 diabetes	0021 (1.8%)	0-	0021 (1.8%)	-	0.730
Gestational diabetes	0211 (18.3%)	002 (11.8%)	0209 (18.4%)	0.59 (0.13–2.60)	0.373

Results are presented as median (range) or absolute frequency (percentage).

*According to the Graffar-Méndez scale [18].

Pregnancy in the 17 GBS colonized women was delivered at term. Nine of these deliveries were vaginal and eutocic. Of the eight abdominal deliveries, four were iterative cesarean section, two for lack of progress in labor, and two for non-reassuring fetal status. Only 6 of the 17 NB of colonized mothers were sampled. None of them was colonized by GBS, and during the three months follow-up, no infectious events attributable to GBS occurred. However, one NB (patient No. 840) had pneumonia associated with birth asphyxia, requiring hospitalization for 20 days without identifying the etiologic microorganism. This NB received ampicillin (150 mg/kg/day) plus amikacin (15 mg/kg/day) without improvement after ten days of treatment. Therapy changed to piperacillin plus tazobactam (150 mg/kg/day piperacillin component) with a favorable evolution. None of the GBS colonized mothers developed infectious complications during the three-month postpartum follow-up. Regarding the serotypes of the 17 GBS isolates, 3 were serotype I, 5 were serotype II, 3 were serotype III, 4 were serotype IV, and 2 were serotype V (Table 2).

Table 2

Clinical and obstetric characteristics of 17 pregnant women colonized by group B Streptococcus and 6 sampled newborns.

Patient No.	Type of delivery	Birth asphyxia	GBS colonization of NB	GBS serotype
18	Eutocic	No	No	Ib
73	Eutocic	No	No	IV
141	Eutocic	No	No	V
204	LPL	No	-	III
210	LPL	No	No	III
414	Iterative	No	-	V
423	Iterative	No	-	Ia
480	Uncertain	Yesa	-	II
610	Eutocic	No	-	II
685	Eutocic	No	-	II
688	Eutocic	No	-	IV
725	Eutocic	No	-	III
755	Eutocic	No	-	IV
833	Uncertain	No	No	II
840	Iterative	Yesb	No	II
865	Iterative	No	-	IV
1038	Eutocic	No	-	Ib

NB: Newborn; LPL: Caesarean section due to lack of progress in labor; Iterative: Iterative cesarean section; Uncertain: Cesarean section due to non-reassuring fetal status.

a Use of balanced general anesthesia during surgery, Apgar 6/7.

b The NB was hospitalized due to pneumonia associated with neonatal asphyxia without the identification of the etiologic microorganism.

The patient was discharged 20 days later after clinical improvement.

Antimicrobial resistance profile

All GBS isolates were susceptible to levofloxacin, moxifloxacin, linezolid, vancomycin, and tigecycline. Otherwise, 11 isolates (65%) were resistant to tetracycline. Isolates of serotypes Ia, II, III, and V were resistant to tetracycline, whereas the isolates of serotypes Ib and IV were susceptible to this antibiotic. Only one isolate was sensitive to all the antibiotics tested (isolate 755 of serotype IV). Concerning clindamycin, seven (41%) of the isolates had intermediate sensitivity. We also identified four isolates with reduced susceptibility to beta-lactam antibiotics: serotype Ib, III, IV, and V. From these later isolates, the serotype Ib and IV also had intermediate susceptibility to clindamycin (Table 3).

Table 3

Level of antibiotic resistance and serotype of 17 group B Streptococcus (GBS) isolated from pregnant women.

		AMP		LEV		MOX		LIN		VAN		TET		TIG		ERY (Disk 15μg)*			CLY (Disk 2μg)*
Range IMC μg ml−1		≤ 0.25	≥ 16	≤ 0.12	≥ 8	≤ 0.25	≥ 8	≤ 0.5	≥ 8	≤ 0.5	≥ 32	≤ 1	≥ 16	≤ 0.12	≥ 2	≥ 21	I 16–20	≤ 15	≥ 19	I 16–18	≤ 15
Isolate No.	Serotype
18	Ib	RS		S		S		S		S		S		S		S			I
73	IV	RS		S		S		S		S		S		S		S			I
141	V	S		S		S		S		S		R		S		S			S
204	III	RS		S		S		S		S		R		S		S			S
210	III	S		S		S		S		S		R		S		S			S
414	V	RS		S		S		S		S		R		S		S			S
423	Ia	S		S		S		S		S		R		S		S			S
480	II	S		S		S		S		S		R		S		S			S
610	II	S		S		S		S		S		R		S		S			S
685	II	S		S		S		S		S		R		S		S			I
688	IV	S		S		S		S		S		S		S		S			I
725	III	S		S		S		S		S		R		S		S			S
755	IV	S		S		S		S		S		S		S		S			S
833	II	S		S		S		S		S		R		S		S			I
840	II	S		S		S		S		S		R		S		S			I
865	IV	S		S		S		S		S		S		S		I			S
1038	Ib	S		S		S		S		S		S		S		S			I

S: Sensible, I: Intermediate, R: Resistant, RS: Reduced susceptibility, AMP: Ampicillin, LEV: Levofloxacin, MOX: Moxifloxacin, LIN: Linezolid, VAN: Vancomycin, TET: Tetracycline, TIG, Tigecycline, ERY: Erythromycin, CLY: Clindamycin.

*Interpretive categories and zone diameter breakpoints (nearest whole mm).

The classification of our isolates was done according to the international expert proposal for interim standard definitions for antimicrobial acquired resistance by Magiorakos AP et al. [22], following the categories and definitions proposed for Enterococcus spp. Seven of our isolates were non-susceptible to one agent in two antimicrobial categories tested, nine isolates were non-susceptible to one agent in one antimicrobial category tested, and only one isolate was susceptible to all antimicrobial categories tested (Table 4). Finally, we had 11 (64.7%) non-susceptible isolates in the tetracyclines category, 4 (23.6%) in the penicillins category, 7 (41.2%) non-susceptible to clindamycin and one non-susceptible to erythromycin (Table 5).

Table 4

Antimicrobial susceptibility and antimicrobial categories of 17 group B Streptococcus.

		Antimicrobials tested
Isolate No.	Serotype	AMP	LEV	MOX	LIN	VAN	TET	TIG	ERY	CLY	No. of NS antimicrobials*	No. of NS categories*
18	Ib	RS	S	S	S	S	S	S	S	I	2	2
73	IV	RS	S	S	S	S	S	S	S	I	2	2
141	V	S	S	S	S	S	R	S	S	S	1	1
204	III	RS	S	S	S	S	R	S	S	S	2	2
210	III	S	S	S	S	S	R	S	S	S	1	1
414	V	RS	S	S	S	S	R	S	S	S	2	2
423	Ia	S	S	S	S	S	R	S	S	S	1	1
480	II	S	S	S	S	S	R	S	S	S	1	1
610	II	S	S	S	S	S	R	S	S	S	1	1
685	II	S	S	S	S	S	R	S	S	I	2	2
688	IV	S	S	S	S	S	S	S	S	I	1	1
725	III	S	S	S	S	S	R	S	S	S	1	1
755	IV	S	S	S	S	S	S	S	S	S	0	0
833	II	S	S	S	S	S	R	S	S	I	2	2
840	II	S	S	S	S	S	R	S	S	I	2	2
865	IV	S	S	S	S	S	S	S	I	S	1	1
1038	Ib	S	S	S	S	S	S	S	S	I	1	1

*Number of antimicrobials to which each isolate was non-susceptible (NS) according to the categories and definitions for Enterococcus spp by Magiorakos AP et al. [22].

S: Sensible, I: Intermediate, R: Resistant, RS: Reduced susceptibility, AMP: Ampicillin, LEV: Levofloxacin, MOX: Moxifloxacin, LIN: Linezolid, VAN: Vancomycin, TET: Tetracycline, TIG, Tigecycline, ERY: Erythromycin, CLY: Clindamycin.

Table 5

Antimicrobial resistance of 17 group B Streptococcus according to the categories and definitions for Enterococcus spp by Magiorakos et al. [22].

Antimicrobial categories	Antimicrobial agents	No. of non-susceptible GBS isolates
Fluoroquinolones	Levofloxacin	0
	Moxifloxacin	0
Glycopeptides	Vancomycin	0
Oxazolidinones	Linezolid	0
Penicillins	Ampicillin	4 (23.6%)
Tetracyclines	Tetracycline	11 (64.7%)
	Erythromycin	1 (5.9%)
	Clindamycin	7 (41.2%)

GBS: group B Streptococcus.

Identification of Cps, virulence and antibiotic resistance genes from whole-genome analysis of GBS isolates

After assembling the complete draft genomes of 14 of the 17 GBS isolates, we conducted a comparative genomic analysis to identify genes encoding Cps, virulence factors, and antibiotic resistance. All data obtained from whole-genome sequencing are deposited in the BioProject and BioSample databases at NCBI with the accession number PRJNA551699. The genes identified in each of the 14 GBS isolates analyzed are described in Table 6. The Cps loci coding for the sialic acid capsular polysaccharide identified in the 14 isolates were Ia, Ib, II, III, IV, and V. We identified two different Cps loci in two isolates: Cps II and III in isolate 204, and Cps II and IV in isolate 865 (Table 6 and Fig 1).

Table 6

Capsular polysaccharide (Cps), virulence factors, and antibiotic resistance genes in group B Streptococcus (GBS) isolated from pregnant women.

Virulence factora	GBS isolate number														Referenceb
	73	141	204	210	414	423	480	610	725	755	833	840	865	1038
Immune evasion
Serotypec	IV	V	III	III	V	Ia	II	II	III	IV	II	II	IV	Ib
Capsular polysaccharide d
Cps Ia	-	-	-	-	-	+	-	-	-	-	-	-	-	-	LT671983.1
Cps Ib	-	-	-	-	-	-	-	-	-	-	-	-	-	+	LT671984.1
Cps II	-	-	+	-	-	-	+	+	-	-	+	+	+	-	LT671985.1
Cps III	-	-	+	+	-	-	-	-	+	-	-	-	-	-	LT671986.1
Cps IV	+	-	-	-	-	-	-	-	-	+	-	-	+	-	LT671987.1
Cps V	-	+	-	-	+	-	-	-	-	-	-	-	-	-	LT671988.1
Cps VI	-	-	-	-	-	-	-	-	-	-	-	-	-	-	LT671989.1
Cps VII	-	-	-	-	-	-	-	-	-	-	-	-	-	-	LT671990.1
Cps VIII	-	-	-	-	-	-	-	-	-	-	-	-	-	-	LT671991.1
Cps IX	-	-	-	-	-	-	-	-	-	-	-	-	-	-	LT671992.1
scpB	+	+	+	+	+	+	+	+	+	+	+	+	+	+	U56908.1
Cell adhesion and invasiveness
lmb	+	+	+	+	+	+	+	+	+	+	+	+	+	+	AF062533.1
fbsA	+	-	+	+	-	+	+	-	+	+	+	+	+	+	AJ437620.1
fbsB	-	-	+	+	-	+	-	-	+	+	-	-	+	-	HQ267707.1
acp	-	-	+	+	+	+	+	-	-	+	+	+	+	+	M97256.1
hvgA	-	-	+	+	-	-	-	-	+	+	-	-	+	-	CP020432.2
Antimicrobial peptide resistance
Pili
PI-1	+	+	+	+	+	+	+	+	+	-	-	+	+	+	EU929743.1
PI-2a	+	+	+	-	+	+	+	+	-	-	+	+	+	+	EU929327.1
PI-2b	-	-	+	+	-	-	-	-	+	+	-	-	+	-	EU929402.1
Pore-forming toxin
cylE	+	+	+	+	+	+	+	+	+	+	+	+	+	+	AF093787.2
Antibiotic resistance
Perfect identitye
mel	-	-	+	-	-	-	-	-	-	-	-	-	-	-
Strict identityf
mprF	+	+	+	+	+	+	+	+	+	+	+	+	+	+
tetM	-	+	+	+	+	-	+	+	+	-	+	+	+	+
mefA	-	-	+	-	-	-	-	-	-	-	-	-	-	-

Classification obtained from Rajagopal L, 2009 [30].

GenBank ID of the sequences used as a reference for gene identification.

Determined by latex agglutination of the capsular polysaccharide-type antigen with the StrepPRO™ Streptococcal Grouping Kit (Hardy Diagnostics; Santa Maria, CA, USA).

Locus Cps mapping by whole genome sequencing.

Detection carried out as clinical surveillance finding 100% coincidences.

Detection of previously reported variants in which mutations may exist finding probably functional genes (https://card.mcmaster.ca/).

Fig 1

Circular map of the Cps loci coding for the sialic acid capsular polysaccharide identified in 14 GBS isolates.

Mapping was made by comparative sequence alignment using the BRIG program. Comparative BLASTn analysis with 70, 90, and 100% identities are displayed, and gaps in circles represent regions with no identity of genes.

We identified ten distinct virulence gene profiles; the most common profile was scpB, lmb, fbsA, acp, PI-1, PI-2a, and cylE (3/14, 21%). The genes scpB, lmb, and cylE were found in the fourteen isolates evaluated, and the acp gene encoding the alpha C protein was found in 10 (71.4%) isolates. The locus for virulence factor PI-1 was second in frequency with 12 positive isolates. PI-2a and fbsA genes were identified in 11 isolates, while gene was found in 10, fbsB gene was found in six isolates, and the loci for factor PI-2b and hvg gene were present in five isolates. Moreover, except for isolates 755 and 833, at least two loci of the Pili virulence factor (PI-1, PI-2a, or PI-2b) were simultaneously found in the genome of each isolate evaluated. Thus, the combination PI-1 + PI-2a was identified in eight isolates, and the combination PI-1 + PI-2b was identified in two. Furthermore, the triple combination of the PI-1 + PI-2a + PI-2b loci was found in isolates 204 and 865. In these two isolates, we identified all the genes coding for virulence factors evaluated (Tables 6 and 7 and Fig 2).

Table 7

Virulence gene profiles of 14 group B Streptococcus isolated from pregnant women.

Virulence Gene Profile	Number of Isolates (%)	Isolates
scpB, lmb, PI-1, PI-2a, cylE	1 (7.1%)	141
scpB, lmb, acp, PI-1, PI-2a, cylE	1 (7.1%)	414
scpB, lmb, fbsA, acp, PI-2a, cylE	1 (7.1%)	833
scpB, lmb, fbsA, PI-1, PI-2a, cylE	2 (14.3%)	73, 610
scpB, lmb, fbsA, acp, PI-1, PI-2a, cylE	3 (21.4%)	480, 840, 1038
scpB, lmb, fbsA, fbsB, acp, PI-1, PI-2a, cylE	1 (7.1%)	423
scpB, lmb, fbsA, fbsB, acp, hvgA, PI-2b, cylE	1 (7.1%)	755
scpB, lmb, fbsA, fbsB, hvgA, PI-1, PI-2b, cylE	1 (7.1%)	725
scpB, lmb, fbsA, fbsB, acp, hvgA, PI-1, PI-2b, cylE	1 (7.1%)	210
scpB, lmb, fbsA, fbsB, acp, hvgA, PI-1, PI-2a, PI-2b, cylE	2 (14.3%)	204, 865

Fig 2

Localization in a circular map of scpB, lmb, fbsA, fbsB, acp, hvgA, Pili (PI-1, PI-2a and PI-2b), and cylE identified in 14 GBS isolates.

Mapping was made by comparative sequence alignment using the BRIG program. Comparative BLASTn analysis with 70, 90 and 100% identities are displayed and gaps in circles represent regions with no identity of genes.

The mprF gene confers resistance to cationic peptides that disrupt the cell membrane, and it was found in all the isolates. The tetM gene associated with resistance to tetracyclines was identified in 11 isolates. While the mel and mefA genes conferring resistance to macrolides was found only in isolate 204 (Table 6).

Discussion

The prevalence of GBS colonization in developed countries ranges from 20% to 30% in pregnant women [31]. In the present study, the prevalence was 1.47% in 1,154 pregnant women from Northeastern Mexico. The highest prevalence of GBS colonization is registered in the Dominican Republic (43.5%) and the lowest in Fiji (2%) and Argentina (1.4%); the latter is similar to our results [32, 33]. A survey carried out in the late 1980s in central Mexico identified GBS cervicovaginal colonization in 13% of 340 pregnant women [34]. While Ocampo-Torres et al., found GBS colonization in 8.6% of pregnant women in a region of southeastern Mexico [35].

In the present study, most women were younger than 30 years, as also were 15 of the 17 GBS colonized women (88.2%). Several studies have demonstrated that children of young mothers (less than 20 years old) have a higher risk of developing GBS early-onset disease [36]. We did not find any newborn with early-onset disease or other complication due to GBS infection, no matter the age of the mother. We found that 23% (4) of the GBS colonized women belonged to a socioeconomic level of relative or critical poverty, but only 12% of the non-colonized women belonged to the same group. A study from Southeastern Mexico classified women according to the degree of marginalization, finding that pregnant women with high and very high socioeconomic marginalization had a prevalence of GBS colonization 1.7 times higher (12.7%) than the other groups [35]. Our results also show that GBS colonization was independent on the number of pregnancies, which contrasts to the report by Anthony et al. in California in 1978, where colonization rates were lower in women after the fourth pregnancy [37].

A systematic review and meta-analysis reported that 48.9% cases of GBS invasive disease in children younger than three months old are caused by serotype III. Although the predominant colonizing serotypes around the world were Ia representing 22.9%, Ib 7.0%, II 6.2%, and V 9.1% [38]. In the USA and Europe, the serotype causing most cases of GBS invasive disease is serotype III [3]. In the present study six serotypes were identified, predominating serotype II, IV, and III. This finding contrasts with studies carried out in central Mexico in the 1980s, in which serotype I predominated (33%) with low participation of serotype III (3%) and a high prevalence of non-typeable isolates (18.2%) [34]. Subsequent studies reported type I as the predominant serotype in central and western Mexico (58.8–61.3%), with a greater number of serotype III isolates (5.9–12.8%) and a lower proportion of non-typeable isolates (0.0–5.9%) than previously reported [39, 40].

A decreasing in the frequency of early-onset neonatal GBS disease has been related to the implementation of screening programs and the use of IAP. A retrospective study in central Mexico showed a higher percentage of sepsis cases and neonatal deaths in children of women who did not receive adequate IAP despite having GBS isolated at the cervicovaginal level or in urine [41]. We did not find transmission of GBS from colonized women to their NB. Moreover, neither mothers nor children had any infectious event attributable to GBS during the three months follow up.

Antibiotic resistance in GBS is of concern due to the role of microorganisms as a leading cause of neonatal disease worldwide [13, 14, 42]. GBS is generally susceptible to beta-lactam antibiotics (including penicillin), the first-line antibiotic for GBS infections and IAP. However, GBS with reduced penicillin susceptibility has been reported more frequently [13]. Resistance to beta-lactam antibiotics in Gram-positive organisms is mainly due to structural changes in the penicillin-binding-proteins (PBPs) caused by acquired mutations in genes that encode PBPs. Instead, resistance to macrolide and lincosamide antibiotics occurs through several mechanisms, including efflux pumps, ribosomal modifications, and drug inactivation. The most widespread resistance mechanism to macrolides is the ribosomal methylation by methyltransferases encoded by erm (erythromycin ribosome methylation) genes. In addition, macrolide efflux (Mef) pumps encoded by the mefA/E gene are also commonly detected. GBS resistance to tetracycline is attributed to the efflux proteins TetK and TetL or to ribosomal protection proteins TetM and TetO. Resistance to vancomycin results from modifications in the glycopeptide target site through the synthesis of peptidoglycan precursors with altered residues that result in a low affinity for this antibiotic [13, 14, 22]. We assessed antibiotic susceptibility by an automated method or disk diffusion and found that 65% of GBS isolates were resistant to tetracycline. Only 1 of the 17 isolates was sensitive to all antibiotics tested. Previous studies have reported a high rate of resistance to tetracycline, this has been correlated with the presence of the tetM and tetK genes [43]. Although tetM seems to be a determinant of GBS tetracycline resistance, some strains carry a nonfunctional tetM conditioning its susceptibility. Even though our isolates are scarce for the proportion of GBS positive to tetM, other studies have also reported isolates with this characteristic. There are GBS isolates resistant to tetracycline without the tetM and tetK, genes, their resistance must be due to other determinants, such as other efflux proteins, ribosomal protection proteins, and enzymatic inactivators [13]. Regarding beta-lactams resistance, we found four strains with reduced susceptibility to beta-lactams. Previous studies have reported that in GBS this characteristic is due to mutations within genes encoding penicillin-biding-proteins (PBP) [13, 44].

Considering that GBS can show high rates of resistance to antibiotics, susceptibility testing of GBS isolated from pregnant women with penicillin allergy is essential for an appropriate drug choice. Furthermore, antibiotic susceptibility testing of all GBS isolated could help to monitor GBS drug resistance [13]. Because IAP is administered to prevent perinatal transmission of GBS, it is also essential to monitor rates of antibiotic resistance. We found a high percentage of resistance in the antimicrobial categories of tetracyclines and penicillins, in addition to non-susceptible isolates to clindamycin and, in one case, to erythromycin. These results are relevant because penicillin is the first-line drug for IAP. Additionally, in cases of a severe allergy to penicillin, clindamycin and erythromycin are recommended as second-line antibiotics in some countries [13, 45]. However, increased resistance to both antibiotics has limited their use. Thus, the Royal College of Obstetricians and Gynecologists (RCOG) no longer recommends the use of clindamycin in the UK, instead recommending vancomycin [46]. Although penicillin remains effective against GBS, increasing reports of isolates with reduced susceptibility are concerning, especially when resistance to second-line antibiotics such as erythromycin and clindamycin remains high among GBS [13, 45].

The comparative genomic analysis of the Cps locus confirmed the serotypes Ia, Ib, II, III, IV, and V previously identified by latex agglutination in our GBS isolates. It is important to notice that the isolates 204 and 865 identified as serotypes III and IV respectively by latex agglutination test showed two Cps each one. Isolate 204 had the Cps II and III and isolate 865 had Cps III and IV. Vagino-rectal co-colonization by multiple GBS serotypes is possible but its detection by standard techniques is complicated, the use of molecular techniques would contribute to a more accurate identification for epidemiological purposes [47].

We identified lmb, scpB, and cylE genes in all isolates, which agree with the study by Udo et al., showing that these three genes are always present in GBS [48]. These genes encode for virulence factors; lmb gene encoding for laminin-binding protein, scpB encoding for C5a peptidase, which is responsible for cleaving the C5a complement factor and cylE encodes the β-hemolysin/cytolysin, a pluripotent toxin associated with the cell surface [49]. The hvgA gene encodes an adhesin related with hypervirulence of GBS isolates and it seems to favor not only neonatal colonization but also the development of invasive disease in newborns. The presence of the hvgA gene has been reported between 15.1% to 55.6% of the isolates [49, 50]. We found it in 36% of our isolates, but none of them caused disease.

The acp gene encoding the surface-associated alpha C protein is responsible for interactions of GBS with epithelial cells and is commonly expressed on the surface of serotypes Ia, Ib, and II [11], we found this gene in most of our isolates, it may be involved in the colonization, but not in causing disease, at least in this study. Fibrinogen-binding protein A (FBPA) encoded by the fbsA gene was found in 78.6% of our isolates and detection of the fbsB gene encoding for the fibrinogen-binding protein B (FBPB) was found only on fbsA positive isolates like Rosenau et al. reported [51]. Two loci code for GBS pili in different regions of the genome designated as pilus islands 1 and 2 (PI-1 and PI-2), the latter presenting two distinct variants, PI-2a and PI-2b [52]. All our GBS isolates had at least one PI gene, the most common combination was PI-1 and PI-2a, but two isolates had all three PI genes. These two isolates were the same having two Cps genes detected. Then, the latter result could be due to co-colonization.

Regarding antibiotic resistance genes, mprF (strict) was found in all isolates as previously reported by LaRock and Nizet [53]. This gene provides resistance to defensins and cationic antimicrobial peptides (CAMPs). We also look for tetM (strict) and mefA (strict) genes involved with tetracyclines resistance. The tetM (strict) gene was identified in 78.6% of our isolates, while Zeng X et al. reported, 83% of 512 GBS isolates. The mefA (strict) gene was found in only 1 isolated (7%), compared to 22 out of 512 (4%) that Zeng et al. identified. Thus, percentages of tetM and mefA genes found in this study were similar to those reported by Zeng X et al. [54]. The mel gene, involved with macrolide resistance, was identified in 1 of our 14 isolates. This gene has been associated with the presence of mefA/E [55]. The isolate 204 (having two Cps) had both genes.

In conclusion, the prevalence of GBS colonization in pregnant women from northeastern Mexico was low (1.47%), and there were no cases of mother-to-child transmission or cases of GBS disease. These results suggest low participation of GBS in perinatal pathology in this population. Consequently, the intentional search for GBS colonization in pregnant women of this region seems not justified, and the indication or not of IAP should be guided only by risk factors. However, follow-up studies with a larger sample number are required to know with certainty the role of GBS in perinatal pathology in this region. Additionally, studies to evaluate if the prevention measures implemented in other countries are also required in Mexico. The analysis of Cps by whole-genome classified the isolates in serotypes Ia, Ib, II, III, IV, or V. These serotypes corresponded to those identified by latex agglutination test, except for two in which two serotypes were found in the same isolate, possibly due to co-colonization by two different GBS. In addition, we detected the genes for virulence factors Lmb, CylE, and ScpB in all our isolates and other virulence genes in variable percentages, among them the major virulence adhesin coded by the hvgA gene. These findings suggest that GBS hypervirulent clones are circulating in the population studied, as previously described in a sample of GBS isolates from Mexico City [56]. Although we did not find evidence of GBS disease, it illustrates the importance of knowing the pathogenic characteristics of GBS populations circulating in different regions. On the other hand, we identified resistance or decreased susceptibility to several antibiotics and antimicrobial categories, including the most commonly used antibiotics in IAP, such as penicillins, clindamycin, and erythromycin. This finding shows the importance of adequately knowing the resistance patterns to prevent and treat perinatal GBS infection.

15 Oct 2021

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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: Comments to authors:

- The current study is interesting; however, the authors should address the following comments to improve the quality of the manuscript:

- The manuscript should be revised for language editing and grammar mistakes.

- Please write the scientific names of bacterial pathogens in a correct form all over the manuscript and the references section (italic):

For example streptococci, it should be Streptococci (S: should be capital not small, and italic)

Title:

I think the work would benefit from the title that contains the main conclusion of the study (should be derived from the conclusion). Please modify the title.

Abstract:

- The abstract must illustrate the used methods and the most prevalent results (give more hints about methods and results). Besides, rephrase the main conclusion of your findings.

Introduction: (it needs to be more informative)

-Give a hint about different infection caused by Group B Streptococci , virulence factors, and the mechanism of disease occurrence.

- The authors should illustrate the public health importance concerning the emergence of multidrug-resistant (MDR) bacterial pathogens that reflecting the necessity of new potent and safe antimicrobial agents. Several studies proved the widespread MDR- bacterial pathogens;

Authors could add the following paragraph:

Multidrug resistance has been increased all over the world that is considered a public health threat. Several recent investigations reported the emergence of multidrug-resistant bacterial pathogens from different origins including humans, birds, cattle, and fish that increase the need for routine application of the antimicrobial susceptibility testing to detect the antibiotic of choice as well as the screening of the emerging MDR strains. You should cite the following valuable studies:

1.PMID: 33177849

2.PMID: 32497922

3.PMID:33061472

4.PMID: 33947875

5.PMID: 32472209

6.PMID: 31170450

7.PMID: 33188216

8.PMID: 30150182

9. PMID: 34445951

-Rephrase the aim of the work to be clear and better sound.

Material and methods

-Isolation and presumptive

- GBS identification and serotyping identification

•Specific references should be added.

•Add the company, city, and country of the used reagents that were used in the biochemical identification of isolates. Also, enumerate all used biochemical reactions.

- Antimicrobial susceptibility testing:

•Illustrate the antimicrobial classes of the tested antimicrobial agents.

•The authors are advised to classify the tested isolates to MDR or XDR as described by Magiorakos et al.

Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, et al. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: An international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect. 2012; 18:268–81. doi:10.1111/j.1469-0691.2011.03570.x.

-Add more data about the used software in the statistical analyses?

-Result:

-Illustrate the phenotypic characteristics of the recovered GBS isolates.

- Line 220: (Antimicrobial susceptibility) should be modified to be:

Antimicrobial resistance profile of the recovered GBS isolates

-Illustrate in a new table the occurrence of MDR (Multidrug resistance) among the recovered isolates (illustrate the names of the antimicrobial classes and different antibiotics):

No. of strains%Type of resistance

R OR MDR OR XDRPhenotypic multidrug resistance

(Antimicrobial classes and different antibiotics).

The antibiotic -resistance genes

- S1 Fig. and S2 Fig. should be placed in the main manuscript.

-Discussion:

- The authors are advised to illustrate the real impact of their findings without repetition of results.

-Illustrate the different mechanisms of antimicrobial resistance in Streptococci.

-Conclusion

- Should be rephrased to be sounded. A real conclusion should focus on the question or claim you articulated in your study, which resolution has been the main objective of your paper?

Reviewer #2: - The current study has a significant impact, but it needs a minor revision:

- The manuscript should be revised for grammar mistakes.

- Please write the scientific names of bacterial pathogens in the correct form all over the manuscript and in the References section (should be italic).

-The title is broad, please modify the title.

- Add more details about the used methods and most prevalent results in the abstract.

-In the introduction: discuss the public health importance of the Group B Streptococci and different infections caused by them.

-Improve the aim of work.

Methods: Good

-Explain the methods of isolation and identification in detail??

-Specific references should be added to all the used methods and techniques.

-Add the manufacturing company, city, and country for the used reagents and antimicrobial discs.

-Results: Good presentation

- Discuss in detail the phenotypic characters of the isolated GBS strains.

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3 Feb 2022

Monterrey, Mexico, February 3, 2022.

Abdelazeem Mohamed Algammal, Prof, Ph.D.

Academic Editor PLOS ONE

Dear Dr. Abdelazeem Mohamed Algammal, Academic Editor of Plos One, this is the rebuttal letter that responds to each point raised by the academic editor and reviewers to the manuscript PONE-D-21-30812: “Genomic analysis of virulence factors and antimicrobial resistance of group B Streptococcus isolated from pregnant women in northeastern Mexico.” We also include a marked-up copy of our manuscript that highlights changes made to the original version and an unmarked version of our revised paper without tracked changes.

Point raised by the academic editor:

• Academic editor comment: If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

Change made or response: The laboratory protocols used in our study have been deposited in protocols.io as described below:

Sample collection procedures are available in protocols.io at dx.doi.org/10.17504/protocols.io.b2iaqcae (lines 120-121).

GBS isolation procedure is available in protocols.io at dx.doi.org/10.17504/protocols.io.b2iaqcae (lines 138-139).

GBS identification and serotyping procedures are available in protocols.io at dx.doi.org/10.17504/protocols.io.b2iaqcae, dx.doi.org/10.17504/protocols.io.b2h7qb9n, dx.doi.org/10.17504/protocols.io.b2ibqcan, dx.doi.org/10.17504/protocols.io.b2h8qb9w, and dx.doi.org/10.17504/protocols.io.b2guqbww, dx.doi.org/10.17504/protocols.io.b2uvqew6 (lines 148-152).

• Academic editor comment: Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

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https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Change made or response: We have ensured that our manuscript meets the PLOS ONE style requirements, including file naming. For this, we have based ourselves on the PLOS ONE style templates of the two electronic addresses provided.

Reviewers' comments:

Reviewer #1: Comments to authors:

- Reviewer Comment: The current study is interesting; however, the authors should address the following comments to improve the quality of the manuscript: - The manuscript should be revised for language editing and grammar mistakes.

Change made or response: The entire manuscript has been revised in its English edition, and grammatical errors have been removed.

- Reviewer Comment: Please write the scientific names of bacterial pathogens in a correct form all over the manuscript and the references section (italic): For example streptococci, it should be Streptococci (S: should be capital not small, and italic)

Change made or response: The scientific names of bacterial pathogens were corrected throughout the manuscript and reference section, as indicated by the reviewer. These changes are highlighted in yellow in the marked copy of our manuscript.

- Reviewer Comment: Title: I think the work would benefit from the title that contains the main conclusion of the study (should be derived from the conclusion). Please modify the title.

Change made or response: The title of the work was modified according to the study's main conclusions as follows: “Genomic analysis of virulence factors and antimicrobial resistance of group B Streptococcus isolated from pregnant women in northeastern Mexico”.

Abstract:

- Reviewer Comment: The abstract must illustrate the used methods and the most prevalent results (give more hints about methods and results). Besides, rephrase the main conclusion of your findings. Introduction: (it needs to be more informative)

Change made or response: In response to the reviewer's recommendations for the abstract, the introduction was made more informative, the methods used were described in more detail, more clues were added about the methods and results of the study, and the main conclusion of the study was rephrased as shown below:

Introduction

The genes lmb, cylE, scpB, and hvgA are involved with increased virulence of GBS, and hypervirulent clones have been identified in different regions. In addition, increasing resistance of GBS to macrolides and lincosamides has been reported, so knowing the patterns of antibiotic resistance may be necessary to prevent and treat GBS infections. This study aimed to identify virulence genes and antibiotic resistance associated with GBS colonization in pregnant women from northeastern Mexico.

Methods

Pregnant women with 35-37 weeks of gestation underwent recto-vaginal swabbing. One swab was inoculated into Todd-Hewitt broth supplemented with gentamicin and nalidixic acid, a second swab was inoculated into LIM enrichment broth, and a third swab was submerged into a transport medium. All samples were subcultured onto blood agar. After overnight incubation, suggestive colonies with or without hemolysis were analyzed to confirm GBS identification by Gram staining, catalase test, hippurate hydrolysis, CAMP test, and incubation in a chromogenic medium. We used latex agglutination to confirm and serotype GBS isolates. Antibiotic resistance patterns were assessed by Vitek 2 and disk diffusion. Periumbilical, rectal and nasopharyngeal swabs were collected from some newborns of colonized mothers. All colonized women and their newborns were followed up for three months to assess the development of disease attributable to GBS. Draft genomes of all GBS isolates were obtained by whole-genome sequencing. In addition, bioinformatic analysis to identify genes encoding capsular polysaccharides and virulence factors was performed using BRIG, while antibiotic resistance genes were identified using the CARD database.

Results

We found 17 GBS colonized women out of 1154 pregnant women (1.47%). None of the six newborns sampled were colonized, and no complications due to GBS were detected in pregnant women or newborns. Three isolates were serotype I, 5 serotype II, 3 serotype III, 4 serotype IV, and 2 serotype V. Ten distinct virulence gene profiles were identified, being scpB, lmb, fbsA, acp, PI-1, PI-2a, cylE the most common (3/14, 21%). The virulence genes identified were scpB, lmb, cylE, PI-1, fbsA, PI-2a, acp, fbsB, PI-2b, and hvgA. We identified resistance to tetracycline in 65% (11/17) of the isolates, intermediate susceptibility to clindamycin in 41% (7/17), and reduced susceptibility to ampicillin in 23.5% (4/17). The tetM gene associated to tetracyclines resistance was found in 79% (11/14) and the mel and mefA genes associated to macrolides resistance in 7% (1/14).

Conclusions

The detection of strains with genes coding virulence factors means that clones with pathogenic potential circulates in this region. On the other hand, the identification of decreased susceptibility to antibiotics from different antimicrobial categories shows the importance of adequately knowing the resistance patterns to prevent and to treat GBS perinatal infection.

- Reviewer Comment: Give a hint about different infection caused by Group B Streptococci, virulence factors, and the mechanism of disease occurrence.

Change made or response: As directed by the reviewer, more information about the different types of GBS infections, their virulence factors, and the mechanisms of disease occurrence have been added to the introduction as shown below:

Group B Streptococcus (GBS; Streptococcus agalactiae) colonizes the human genitourinary and gastrointestinal tract and causes a variety of infectious processes in pregnant women, such as asymptomatic bacteriuria, urinary tract infection, bacteremia, pneumonia, meningitis, endocarditis, sepsis, and various obstetric complications as spontaneous abortion, premature labor, chorioamnionitis, endometritis, stillbirth, and neonatal and maternal death. In addition, according to the onset of clinical manifestations, GBS infection in newborns (NB) can present with bacteremia, pneumonia, and sepsis as early-onset disease, late-onset disease, or late-late-onset disease [1, 2]. (Line 81-88)

Previous studies reported several factors making GBS more virulent and resistant to antibiotics. The most studied virulence factor is the capsular polysaccharide (Cps), which defines GBS serotypes (Ia, Ib, II-IX) and contributes to evade the immune system. However, other factors, such as laminin-binding protein (Lmb), fibrinogens (Fbs), hypervirulent adhesin (HvgA), and alpha-C protein (ACP), are associated with adherence and cell invasion [9, 10, 11]. In addition, an increasing GBS resistance to macrolides, lincosamides, and tetracyclines has been reported by several authors worldwide [12, 13]. (Line 98-104)

- Reviewer Comment: The authors should illustrate the public health importance concerning the emergence of multidrug-resistant (MDR) bacterial pathogens that reflecting the necessity of new potent and safe antimicrobial agents. Several studies proved the widespread MDR- bacterial pathogens;

Authors could add the following paragraph:

Multidrug resistance has been increased all over the world that is considered a public health threat. Several recent investigations reported the emergence of multidrug-resistant bacterial pathogens from different origins including humans, birds, cattle, and fish that increase the need for routine application of the antimicrobial susceptibility testing to detect the antibiotic of choice as well as the screening of the emerging MDR strains. You should cite the following valuable studies:

1.PMID: 33177849

2.PMID: 32497922

3.PMID:33061472

4.PMID: 33947875

5.PMID: 32472209

6.PMID: 31170450

7.PMID: 33188216

8.PMID: 30150182

9.PMID: 34445951

Change made or response: In response to this reviewer's recommendation, we added the following statement at the end of the introduction and included several of the references suggested by this reviewer:

Multidrug resistance has been increased all over the world that is considered a public health threat. Several recent investigations reported the emergence of multidrug-resistant bacterial pathogens from different origins, including humans, birds, cattle, and fish, that increase the need for routine application of antimicrobial susceptibility testing to detect the antibiotic of choice and the screening of the emerging MDR strains [13-17]. (Line 104-108)

- Reviewer Comment: Rephrase the aim of the work to be clear and better sound.

Change made or response: The aim of the study was rephrased as follows:

The aim of the present study was to explore the presence of virulence and antibiotic resistance genes in GBS associated with colonization in pregnant women in a population from northeastern Mexico. (Line 108-110)

Material and methods

- Reviewer Comment: Isolation and presumptive GBS identification and serotyping identification

•Specific references should be added.

Change made or response: The laboratory protocols used in our study were deposited in protocols.io as described below:

Sample collection procedures are available in protocols.io at dx.doi.org/10.17504/protocols.io.b2iaqcae (Line 120-121).

GBS isolation procedure used is available in protocols.io at dx.doi.org/10.17504/protocols.io.b2iaqcae (Line 138-139).

GBS identification and serotyping procedures are available in protocols.io at dx.doi.org/10.17504/protocols.io.b2iaqcae, dx.doi.org/10.17504/protocols.io.b2h7qb9n, dx.doi.org/10.17504/protocols.io.b2ibqcan, dx.doi.org/10.17504/protocols.io.b2h8qb9w, dx.doi.org/10.17504/protocols.io.b2guqbww, and dx.doi.org/10.17504/protocols.io.b2uvqew6 (Line 148-152).

In addition, we added the following references to support the isolation, identification, and serotyping methods used:

19. Filkins L, Hauser J, Robinson-Dunn B, Tibbetts R, Boyanton B, Revell P, on behalf of the American Society for Microbiology Clinical and Public Health Microbiology Committee, Subcommittee on Laboratory Practices. 2020. Guidelines for detection and identification of group B Streptococcus. American Society for Microbiology. https://asm.org/Guideline/Guidelines-for-the-Detection-and-Identification-of.

20. Hansen SM, Sørensen UB. Method for quantitative detection and presumptive identification of group B Streptococci on primary plating. J Clin Microbiol. 2003 Apr;41(4):1399-403. doi: 10.1128/JCM.41.4.1399-1403.2003.

21. Slotved HC, Elliott J, Thompson T, Konradsen HB. Latex assay for serotyping of group B Streptococcus isolates. J Clin Microbiol. 2003;41(9):4445-7. doi: 10.1128

- Reviewer Comment: Add the company, city, and country of the used reagents that were used in the biochemical identification of isolates. Also, enumerate all used biochemical reactions.

Change made or response: We added the company, city, and country of all reagents used in the biochemical identification of the isolates, and all the biochemical reactions used were listed. (Lines 132, 170, 172, 175, and 176)

- Reviewer Comment: Antimicrobial susceptibility testing:

• Illustrate the antimicrobial classes of the tested antimicrobial agents.

•The authors are advised to classify the tested isolates to MDR or XDR as described by Magiorakos et al.

Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, et al. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: An international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect. 2012; 18:268–81. doi:10.1111/j.1469-0691.2011.03570.x.

Change made or response: The different classes of antimicrobials tested were added as a list, as shown below:

The categories of antimicrobials tested were fluoroquinolones, glycopeptides, glycylcyclines, oxazolidinones, penicillins, tetracyclines, in addition to erythromycin and clindamycin [22]. (Lines 155 and 156)

We also added the classification of GBS isolates based on the definition by Magiorakos et al. in the results section.

- Reviewer Comment: Add more data about the used software in the statistical analyses?

Change made or response Change made or response: We completed the information of the software used in the statistical analysis as shown below:

The analysis was made using the IBM SPSS Statistics software (Version 20; SPSS Inc., Armonk, NY, USA). (Lines 196 and 197)

- Result:

- Reviewer Comment: Illustrate the phenotypic characteristics of the recovered GBS isolates.

- Change made or response:

Phenotypic characteristics of GBS isolates are shown in different parts of the text and tables in the results section and include serotype and antimicrobial resistance profiles. (Lines 232-279)

The genotypic characteristics from the genome-wide analysis are also described in the text, tables, and figures. (Lines 281-337)

- Reviewer Comment: Line 220: (Antimicrobial susceptibility) should be modified to be: Antimicrobial resistance profile of the recovered GBS isolates

Change made or response: Based on this recommendation, we changed the subtitle of this section to read “Antimicrobial resistance profile”. (Line 246)

- Reviewer Comment: Illustrate in a new table the occurrence of MDR (Multidrug resistance) among the recovered isolates (illustrate the names of the antimicrobial classes and different antibiotics):

No. of strains%Type of resistance

R OR MDR OR XDRPhenotypic multidrug resistance

(Antimicrobial classes and different antibiotics).

The antibiotic -resistance genes

Change made or response: In this new version of our manuscript, the information on antimicrobial resistance is presented in four tables.

• Table 3 describes the level of antibiotic resistance and serotype of GBS isolates. (Lines 255-258)

• Table 4 presents the antimicrobial susceptibility, along with the number of antimicrobials and the number of antimicrobial categories to which the isolates showed resistance or decreased susceptibility according to the classification by Magiorakos et al. (Lines 268-273)

• Table 5 shows the antimicrobial resistance of GBS isolates classified by agents and antimicrobial categories according to Magiorakos et al. (Lines 276-279)

• The information on identifying genes related to antimicrobial resistance is included in Table 6. (Lines 324-333).

- Reviewer Comment: S1 Fig. and S2 Fig. should be placed in the main manuscript.

Change made or response: Figure 1 and Figure 2 are part of the main manuscript in this new version. (Lines 292-296 and 312-316)

- Discussion:

- Reviewer Comment: The authors are advised to illustrate the real impact of their findings without repetition of results.

Change made or response: To illustrate the real impact of our findings, we added the following paragraphs in the discussion section:

• Because IAP is administered to prevent perinatal transmission of GBS, it is also essential to monitor rates of antibiotic resistance. We found a high percentage of resistance in the antimicrobial categories of tetracyclines and penicillins, in addition to non-susceptible isolates to clindamycin and, in one case, to erythromycin. These results are relevant because penicillin is the first-line drug for IAP. Additionally, in cases of a severe allergy to penicillin, clindamycin and erythromycin are recommended as second-line antibiotics in some countries [13, 45]. However, increased resistance to both antibiotics has limited their use. Thus, the Royal College of Obstetricians and Gynecologists (RCOG) no longer recommends the use of clindamycin in the UK, instead recommending vancomycin [46]. Although penicillin remains effective against GBS, increasing reports of isolates with reduced susceptibility are concerning, especially when resistance to second-line antibiotics such as erythromycin and clindamycin remains high among GBS [13, 45]. (Lines 410-422)

• In addition, we detected the genes for virulence factors Lmb, CylE, and ScpB in all our isolates and other virulence genes in variable percentages, among them the major virulence adhesin coded by the hvgA gene. These findings suggest that GBS hypervirulent clones are circulating in the population studied, as previously described in a sample of GBS isolates from Mexico City [56]. Although we did not find evidence of GBS disease, they illustrate the importance of knowing the pathogenic characteristics of GBS populations circulating in different regions. On the other hand, we identified resistance or decreased susceptibility to several antibiotics and antimicrobial categories, including the most commonly used antibiotics in IAP, such as penicillins, clindamycin, and erythromycin. This finding shows the importance of adequately knowing the resistance patterns to prevent and treat perinatal GBS infection. (Lines 473-483)

- Reviewer Comment: Illustrate the different mechanisms of antimicrobial resistance in Streptococci.

Change made or response: To illustrate the different mechanisms of antimicrobial resistance in GBS, we also add the following paragraph:

• Antibiotic resistance in GBS is of concern due to the role of microorganisms as a leading cause of neonatal disease worldwide [13, 14, 42]. GBS is generally susceptible to beta-lactam antibiotics (including penicillin), the first-line antibiotic for GBS infections and IAP. However, GBS with reduced penicillin susceptibility has been reported more frequently [13]. Resistance to beta-lactam antibiotics in Gram-positive organisms is mainly due to structural changes in the penicillin-binding-proteins (PBPs) caused by acquired mutations in genes that encode PBPs. Instead, resistance to macrolide and lincosamide antibiotics occurs through several mechanisms, including efflux pumps, ribosomal modifications, and drug inactivation. The most widespread resistance mechanism to macrolides is the ribosomal methylation by methyltransferases encoded by erm (erythromycin ribosome methylation) genes. In addition, macrolide efflux (Mef) pumps encoded by the mefA/E gene are also commonly detected. GBS resistance to tetracycline is attributed to the efflux proteins TetK and TetL or to ribosomal protection proteins TetM and TetO. Resistance to vancomycin results from modifications in the glycopeptide target site through the synthesis of peptidoglycan precursors with altered residues that result in a low affinity for this antibiotic [13, 14, 22]. (Lines 378-393)

- Conclusion

- Reviewer Comment: Should be rephrased to be sounded. A real conclusion should focus on the question or claim you articulated in your study, which resolution has been the main objective of your paper?

Change made or response: To focus the conclusion on the objective and main findings of the study, we added the following information in the conclusion paragraph:

• In addition, we detected the genes for virulence factors Lmb, CylE, and ScpB in all our isolates and other virulence genes in variable percentages, among them the major virulence adhesin coded by the hvgA gene. These findings suggest that GBS hypervirulent clones are circulating in the population studied, as previously described in a sample of GBS isolates from Mexico City [56]. Although we did not find evidence of GBS disease, they illustrate the importance of knowing the pathogenic characteristics of GBS populations circulating in different regions. On the other hand, we identified resistance or decreased susceptibility to several antibiotics and antimicrobial categories, including the most commonly used antibiotics in IAP, such as penicillins, clindamycin, and erythromycin. This finding shows the importance of adequately knowing the resistance patterns to prevent and treat perinatal GBS infection. (Lines 473-483)

Reviewer #2: The current study has a significant impact, but it needs a minor revision:

- Reviewer Comment: The manuscript should be revised for grammar mistakes.

Change made or response: The whole manuscript has been revised in its English edition, and grammatical errors have been removed.

- Reviewer Comment: Please write the scientific names of bacterial pathogens in the correct form all over the manuscript and in the References section (should be italic).

Change made or response: The scientific names of bacterial pathogens were corrected throughout the manuscript and reference section, as indicated by the reviewer. These changes are highlighted in yellow in the marked copy of our manuscript.

- Reviewer Comment: The title is broad, please modify the title.

Change made or response: The work title was modified according to the study's main conclusions as follows: “Genomic analysis of virulence factors and antimicrobial resistance of group B Streptococcus isolated from pregnant women in northeastern Mexico”.

- Reviewer Comment: Add more details about the used methods and most prevalent results in the abstract.

Change made or response: Within the abstract introduction was made more informative, the methods used were described in more detail, more clues were added about the methods and results, and the main conclusion of the study was rephrased as shown below:

Introduction

The genes lmb, cylE, scpB, and hvgA are involved with increased virulence of GBS, and hypervirulent clones have been identified in different regions. In addition, increasing resistance of GBS to macrolides and lincosamides has been reported, so knowing the patterns of antibiotic resistance may be necessary to prevent and treat GBS infections. This study aimed to identify virulence genes and antibiotic resistance associated with GBS colonization in pregnant women from northeastern Mexico.

Methods

Pregnant women with 35-37 weeks of gestation underwent recto-vaginal swabbing. One swab was inoculated into Todd-Hewitt broth supplemented with gentamicin and nalidixic acid, a second swab was inoculated into LIM enrichment broth, and a third swab was submerged into a transport medium. All samples were subcultured onto blood agar. After overnight incubation, suggestive colonies with or without hemolysis were analyzed to confirm GBS identification by Gram staining, catalase test, hippurate hydrolysis, CAMP test, and incubation in a chromogenic medium. We used latex agglutination to confirm and serotype GBS isolates. Antibiotic resistance patterns were assessed by Vitek 2 and disk diffusion. Periumbilical, rectal and nasopharyngeal swabs were collected from some newborns of colonized mothers. All colonized women and their newborns were followed up for three months to assess the development of disease attributable to GBS. Draft genomes of all GBS isolates were obtained by whole-genome sequencing. In addition, bioinformatic analysis to identify genes encoding capsular polysaccharides and virulence factors was performed using BRIG, while antibiotic resistance genes were identified using the CARD database.

Results

We found 17 GBS colonized women out of 1154 pregnant women (1.47%). None of the six newborns sampled were colonized, and no complications due to GBS were detected in pregnant women or newborns. Three isolates were serotype I, 5 serotype II, 3 serotype III, 4 serotype IV, and 2 serotype V. Ten distinct virulence gene profiles were identified, being scpB, lmb, fbsA, acp, PI-1, PI-2a, cylE the most common (3/14, 21%). The virulence genes identified were scpB, lmb, cylE, PI-1, fbsA, PI-2a, acp, fbsB, PI-2b, and hvgA. We identified resistance to tetracycline in 65% (11/17) of the isolates, intermediate susceptibility to clindamycin in 41% (7/17), and reduced susceptibility to ampicillin in 23.5% (4/17). The tetM gene associated to tetracyclines resistance was found in 79% (11/14) and the mel and mefA genes associated to macrolides resistance in 7% (1/14).

Conclusions

The detection of strains with genes coding virulence factors means that clones with pathogenic potential circulates in this region. On the other hand, the identification of decreased susceptibility to antibiotics from different antimicrobial categories shows the importance of adequately knowing the resistance patterns to prevent and to treat GBS perinatal infection.

- Reviewer Comment: In the introduction: discuss the public health importance of the Group B Streptococci and different infections caused by them.

Change made or response: We added more information in the introduction section about the different types of GBS infections, virulence factors, and mechanisms of disease as shown below:

• Group B Streptococcus (GBS; Streptococcus agalactiae) colonizes the human genitourinary and gastrointestinal tract and causes a variety of infectious processes in pregnant women, such as asymptomatic bacteriuria, urinary tract infection, bacteremia, pneumonia, meningitis, endocarditis, sepsis, and various obstetric complications as spontaneous abortion, premature labor, chorioamnionitis, endometritis, stillbirth, and neonatal and maternal death. In addition, according to the onset of clinical manifestations, GBS infection in newborns (NB) can present with bacteremia, pneumonia, and sepsis as early-onset disease, late-onset disease, or late-late-onset disease [1, 2]. (Line 81-88)

• Previous studies reported several factors making GBS more virulent and resistant to antibiotics. The most studied virulence factor is the capsular polysaccharide (Cps), which defines GBS serotypes (Ia, Ib, II-IX) and contributes to evade the immune system. However, other factors, such as laminin-binding protein (Lmb), fibrinogens (Fbs), hypervirulent adhesin (HcgA), and alpha-C protein (ACP), are associated with adherence and cell invasion [9, 10, 11]. In addition, an increasing GBS resistance to macrolides, lincosamides, and tetracyclines has been reported by several authors worldwide [12, 13]. (Line 98-104)

• Multidrug resistance has been increased all over the world that is considered a public health threat. Several recent investigations reported the emergence of multidrug-resistant bacterial pathogens from different origins, including humans, birds, cattle, and fish, that increase the need for routine application of antimicrobial susceptibility testing to detect the antibiotic of choice and the screening of the emerging MDR strains [13-17]. (Line 104-108)

- Reviewer Comment: Improve the aim of work.

Change made or response: The aim of the study was rephrased as follows:

The aim of the present study was to explore the presence of virulence and antibiotic resistance genes in GBS associated with colonization in pregnant women in a population from northeastern Mexico. (Line 108-110)

Methods: Good

- Reviewer Comment: Explain the methods of isolation and identification in detail??

Change made or response: The laboratory protocols used in our study were deposited and are available in protocols.io as described below:

Sample collection procedures are available in protocols.io at dx.doi.org/10.17504/protocols.io.b2iaqcae (Line 120-121).

GBS isolation procedure used is available in protocols.io at dx.doi.org/10.17504/protocols.io.b2iaqcae (Line 138-139).

GBS identification and serotyping procedures are available in protocols.io at dx.doi.org/10.17504/protocols.io.b2iaqcae, dx.doi.org/10.17504/protocols.io.b2h7qb9n, dx.doi.org/10.17504/protocols.io.b2ibqcan, dx.doi.org/10.17504/protocols.io.b2h8qb9w, dx.doi.org/10.17504/protocols.io.b2guqbww, and dx.doi.org/10.17504/protocols.io.b2uvqew6 (Line 148-152).

The different classes of antimicrobials tested were added as a list, as follows:

The categories of antimicrobials tested were fluoroquinolones, glycopeptides, glycylcyclines, oxazolidinones, penicillins, tetracyclines, in addition to erythromycin and clindamycin [22]. (Lines 154 and 156)

Additionally, we added the classification of GBS isolates based on the definition by Magiorakos et al. in the results section.

We also completed the information of the software used in the statistical analysis as follows: The analysis was made using the IBM SPSS Statistics software (Version 20; SPSS Inc., Armonk, NY, USA). (Lines 196 and 197)

- Specific references should be added to all the used methods and techniques.

Change made or response: We added the following references to support the isolation, identification, and serotyping methods used:

19. Filkins L, Hauser J, Robinson-Dunn B, Tibbetts R, Boyanton B, Revell P, on behalf of the American Society for Microbiology Clinical and Public Health Microbiology Committee, Subcommittee on Laboratory Practices. 2020. Guidelines for detection and identification of group B Streptococcus. American Society for Microbiology. https://asm.org/Guideline/Guidelines-for-the-Detection-and-Identification-of.

20. Hansen SM, Sørensen UB. Method for quantitative detection and presumptive identification of group B streptococci on primary plating. J Clin Microbiol. 2003 Apr;41(4):1399-403. doi: 10.1128/JCM.41.4.1399-1403.2003.

21. Slotved HC, Elliott J, Thompson T, Konradsen HB. Latex assay for serotyping of group B Streptococcus isolates. J Clin Microbiol. 2003;41(9):4445-7. doi: 10.1128

- Reviewer Comment: Add the manufacturing company, city, and country for the used reagents and antimicrobial discs.

Change made or response: We added the company, city, and country of all reagents used in the biochemical identification of the isolates, and all the biochemical reactions used were listed. (Lines 132, 170, 172, 175, and 176)

- Reviewer Comment: Results: Good presentation

Change made or response: Thank you very much for your comment.

- Reviewer Comment: Discuss in detail the phenotypic characters of the isolated GBS strains.

Change made or response:

Phenotypic characteristics of GBS isolates are shown in different parts of the text and tables in the results section and include serotype and antimicrobial resistance profiles. (Lines 232-279)

The genotypic characteristics from the genome-wide analysis are also described in the text, tables, and figures. (Lines 281-337)

- Reviewer Comment: Please, place the supplementary figures in the main manuscript.

Change made or response: Figures 1 and Figure 2 are part of the main manuscript in this new version. (Lines 292-296 and 312-316)

- Discussion:

- Reviewer Comment: Please improve (Avoid repetition of results)

Change made or response: We added the following paragraphs discussing our phenotypic and genotypic findings related to antibiotic resistance:

• Because IAP is administered to prevent perinatal transmission of GBS, it is also essential to monitor rates of antibiotic resistance. We found a high percentage of resistance in the antimicrobial categories of tetracyclines and penicillins, in addition to non-susceptible isolates to clindamycin and, in one case, to erythromycin. These results are relevant because penicillin is the first-line drug for IAP. Additionally, in cases of a severe allergy to penicillin, clindamycin and erythromycin are recommended as second-line antibiotics in some countries [13, 45]. However, increased resistance to both antibiotics has limited their use. Thus, the Royal College of Obstetricians and Gynecologists (RCOG) no longer recommends the use of clindamycin in the UK, instead recommending vancomycin [46]. Although penicillin remains effective against GBS, increasing reports of isolates with reduced susceptibility are concerning, especially when resistance to second-line antibiotics such as erythromycin and clindamycin remains high among GBS [13, 45]. (Lines 410-422)

• In addition, we detected the genes for virulence factors Lmb, CylE, and ScpB in all our isolates and other virulence genes in variable percentages, among them the major virulence adhesin coded by the hvgA gene. These findings suggest that GBS hypervirulent clones are circulating in the population studied, as previously described in a sample of GBS isolates from Mexico City [56]. Although we did not find evidence of GBS disease, they illustrate the importance of knowing the pathogenic characteristics of GBS populations circulating in different regions. On the other hand, we identified resistance or decreased susceptibility to several antibiotics and antimicrobial categories, including the most commonly used antibiotics in IAP, such as penicillins, clindamycin, and erythromycin. This finding shows the importance of adequately knowing the resistance patterns to prevent and treat perinatal GBS infection. (Lines 473-483)

• Antibiotic resistance in GBS is of concern due to the role of microorganisms as a leading cause of neonatal disease worldwide [13, 14, 42]. GBS is generally susceptible to beta-lactam antibiotics (including penicillin), the first-line antibiotic for GBS infections and IAP. However, GBS with reduced penicillin susceptibility has been reported more frequently [13]. Resistance to beta-lactam antibiotics in Gram-positive organisms is mainly due to structural changes in the penicillin-binding-proteins (PBPs) caused by acquired mutations in genes that encode PBPs. Instead, resistance to macrolide and lincosamide antibiotics occurs through several mechanisms, including efflux pumps, ribosomal modifications, and drug inactivation. The most widespread resistance mechanism to macrolides is the ribosomal methylation by methyltransferases encoded by erm (erythromycin ribosome methylation) genes. In addition, macrolide efflux (Mef) pumps encoded by the mefA/E gene are also commonly detected. GBS resistance to tetracycline is attributed to the efflux proteins TetK and TetL or to ribosomal protection proteins TetM and TetO. Resistance to vancomycin results from modifications in the glycopeptide target site through the synthesis of peptidoglycan precursors with altered residues that result in a low affinity for this antibiotic [13, 14, 22]. (Lines 378-393)

- Reviewer Comment: Please improve the main conclusion of the manuscript.

Change made or response: To focus the conclusion on the objective and main findings of the study, we added the following information in the conclusion paragraph:

• In addition, we detected the genes for virulence factors Lmb, CylE, and ScpB in all our isolates and other virulence genes in variable percentages, among them the major virulence adhesin coded by the hvgA gene. These findings suggest that GBS hypervirulent clones are circulating in the population studied, as previously described in a sample of GBS isolates from Mexico City [56]. Although we did not find evidence of GBS disease, they illustrate the importance of knowing the pathogenic characteristics of GBS populations circulating in different regions. On the other hand, we identified resistance or decreased susceptibility to several antibiotics and antimicrobial categories, including the most commonly used antibiotics in IAP, such as penicillins, clindamycin, and erythromycin. This finding shows the importance of adequately knowing the resistance patterns to prevent and treat perinatal GBS infection. (Lines 473-483)

6. 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.

Response: We agree that the peer review history of our article is published, including the full peer review and any attached files.

We have considered all the suggestions made by the reviewers, and the changes made are highlighted in yellow within the text of one of the files we uploaded. We hope that the modifications introduced may be appropriate and that our manuscript may be considered for publication in Plos One, and we look forward to a prompt response. Many thanks for all your attention.

Dr. Gerardo del Carmen Palacios Saucedo

Main author

Submitted filename: Response to Reviewers.pdf

Click here for additional data file.

8 Feb 2022

Genomic analysis of virulence factors and antimicrobial resistance of group B Streptococcus isolated from pregnant women in northeastern Mexico

PONE-D-21-30812R1

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Reviewer #1: All comments have been addressed

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Reviewer #1: The authors have carried out significant changes to the manuscript. They have addressed all the suggested corrections and comments. Really, it's an interesting study that has a significant impact. Now, the manuscript could be accepted.

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5 Mar 2022

PONE-D-21-30812R1

Genomic analysis of virulence factors and antimicrobial resistance of group B Streptococcus isolated from pregnant women in northeastern Mexico

Dear Dr. Rivera-Morales:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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