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
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.

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.  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  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 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.

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.  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][14][15][16][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) 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) 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)