Introduction

Streptococcus agalactiae or group B streptococcus (GBS) is a gram-positive bacterium that is present in 10-30% of pregnant women in the United States. Maternal GBS colonization during pregnancy can result in the vertical transmission of the bacterium to infants during the intrapartum period, leading to neonatal sepsis, pneumonia, and meningitis. Additionally, GBS infections can cause urinary tract infections, arthritis, and pneumonia in adults.

Current recommendations endorse universal screening for vaginal and rectal GBS colonization in all pregnant women at 35-37 weeks of gestation. In addition, antibiotic prophylaxis is recommended during pregnancy for those at risk. These measures have led to a decrease in the incidence of invasive early-onset GBS disease in neonates from about 1.7 per 1000 live births to approximately 0.3 cases per 1000 live births. However, GBS remains the leading infectious cause of morbidity and mortality among infants in the United States, and there is a continued need to develop novel prevention and screening protocols.

An understanding of the mechanisms responsible for GBS persistence in the genital tract as well as identification of host defenses employed to combat its presence are pivotal to our efforts to minimize maternal GBS colonization and prevent the acquisition of neonatal infections, but remain incompletely identified.

Recent reports have identified autophagy as a major contributor to the control of intracellular bacterial infections. Autophagy is a basic survival process present in all cells that sequesters and removes defective or aged mitochondria and inflammasomes, dysfunctional protein aggregates, and microorganisms from the cytoplasm. The targeted components are engulfed within a double membrane structure known as the autophagosome. Subsequent fusion of the autophagosome with a lysosome results in degradation of the engulfed component by lysosomal acid hydrolases. The breakdown products–amino acids, nucleotides, fatty acids, and simple carbohydrates–are transported back into the cytoplasm for reutilization. GBS strains have been shown to invade vaginal epithelial cells and survive within the intracellular environment, and activation of autophagy as a consequence of GBS infection has been demonstrated in vitro. However, it remains unclear if GBS modulates autophagy within cells present in the female genital tract.

In addition to autophagy there is a second major mechanism induced when cells are under physiological stress, ie, activation of the inducible 70-kDa heat shock protein (hsp70). In contrast to autophagy whose function is to rid the cells of defective components including microbial antigens, hsp70 binds to intracellular proteins and preserves their conformation and functional capacity. Hsp70 induction has been shown to down-regulate autophagy, by activating mammalian target of rapamycin complex 1, the major inhibitor of autophagy. In addition, mammalian target of rapamycin complex 1 activates heat shock factor 1, the transcription factor that induces transcription of the gene coding for hsp70.

The objective of this study was to identify differences in the extent of autophagy as well as in the concentration of biomarkers previously shown to be involved in vaginal antimicrobial innate immunity between GBS-positive and -negative women.

Materials and Methods

In this prospective cohort study healthy pregnant women were recruited from the outpatient clinic of the department of obstetrics and gynecology at Weill Cornell Medicine from September 2013 through June 2015. All pregnant women between 18-50 years old with a singleton gestation were eligible for inclusion. Exclusion criteria included multifetal pregnancy, preexisting diabetes mellitus, chronic hypertension, cardiac disease, and signs and symptoms of an active infection. Demographic and medical history information were collected from the institutional electronic medical records. Patients were subsequently classified into 2 groups based on the presence or absence of GBS. The GBS-positive group was composed of women in which GBS was detected by a rectovaginal culture at 35-37 weeks of gestation, as well as those where GBS was isolated from the urine at any time during pregnancy. GBS specimens were collected according to published guidelines. The study was approved by the institutional review board at Weill Cornell Medicine and complied with the ethical principles for medical research as outlined in the Declaration of Helsinki. All participants provided informed written consent.

A sample was obtained by vigorously rubbing a cotton swab against the posterior vaginal fornix of each participant at 35-37 weeks of gestation, just prior to the standard GBS rectovaginal sample collection. The samples obtained thus were composed of secretions, exfoliated epithelial cells, as well as cells that were present on the outermost epithelial layers. The contents of the swabs were released into tubes containing 1 mL of sterile phosphate-buffered saline by vigorous agitation. Samples were centrifuged, and supernatant and cell pellet fractions were collected and stored separately at –80°C until used for analysis.

The vaginal supernatant was assayed for matrix metalloproteinase (MMP)-8, neutrophil gelatinase-associated lipocalin (NGAL), hyaluronan, extracellular MMP inducer (EMMPRIN) (R&D Systems, Minneapolis, MN), and alpha amylase (AbCam, Cambridge, United Kingdom) using commercial enzyme-linked immunosorbent assay kits. Concentrations of D- and L-lactic acid were determined colorimetrically using a commercial kit (BioAssay Systems, Hayward, CA). The total protein content of each vaginal supernatant was determined using a commercial colorimetric assay (Pierce, Rockford, IL). The lower limits of sensitivity were: 78 pg/mL for NGAL, 39 pg/mL for EMMPRIN, 62.5 pg/mL for MMP-8, 0.4 ng/mL for hyaluronan, 0.3 mU/mL for amylase, and 0.02 mmol/L for D- and L-lactic acid. Values of MMP-8, NGAL, hyaluronan, and EMMPRIN were converted to ng/μg or pg/μg total protein and mU/ug for amylase.

The cellular fraction was resuspended and lysed in a detergent-based buffer containing protease inhibitors, as described previously. The lysate was then centrifuged and the supernatant collected and immediately assayed for p62 protein and hsp70 using commercial enzyme-linked immunosorbent assay kits (R&D Systems, Minneapolis, MN/Enzo Life Sciences, Farmingdale, NY). p62 (also known as sequestsome-1) is a protein in the cytoplasm that binds to intracellular compounds that are destined to be engulfed by autophagosome and degraded. The p62 is simultaneously degraded and so the concentration of p62 that is detectable in the cytoplasm is inversely proportional to the extent of autophagy induction. The lower limit of sensitivity was 16 pg/mL for hsp70 and 100 pg/mL for p62. Laboratory personnel were blinded to all clinical data.

Statistical analysis was performed using GraphPad (Graphpad Software Inc, San Diego, CA) and SPSS v22 (IBM Corp, Armonk, NY). Categorical variables were evaluated using χ ² analysis or Fisher exact test for variables with low frequencies. Associations between the assays and GBS status were evaluated by the nonparametric Mann-Whitney U test since the values were not normally distributed. Correlations between the different mediators were examined by the Spearman rank correlation test. All P values were 2-sided, with alpha level of statistical significance set at .05.

Materials and Methods

In this prospective cohort study healthy pregnant women were recruited from the outpatient clinic of the department of obstetrics and gynecology at Weill Cornell Medicine from September 2013 through June 2015. All pregnant women between 18-50 years old with a singleton gestation were eligible for inclusion. Exclusion criteria included multifetal pregnancy, preexisting diabetes mellitus, chronic hypertension, cardiac disease, and signs and symptoms of an active infection. Demographic and medical history information were collected from the institutional electronic medical records. Patients were subsequently classified into 2 groups based on the presence or absence of GBS. The GBS-positive group was composed of women in which GBS was detected by a rectovaginal culture at 35-37 weeks of gestation, as well as those where GBS was isolated from the urine at any time during pregnancy. GBS specimens were collected according to published guidelines. The study was approved by the institutional review board at Weill Cornell Medicine and complied with the ethical principles for medical research as outlined in the Declaration of Helsinki. All participants provided informed written consent.

A sample was obtained by vigorously rubbing a cotton swab against the posterior vaginal fornix of each participant at 35-37 weeks of gestation, just prior to the standard GBS rectovaginal sample collection. The samples obtained thus were composed of secretions, exfoliated epithelial cells, as well as cells that were present on the outermost epithelial layers. The contents of the swabs were released into tubes containing 1 mL of sterile phosphate-buffered saline by vigorous agitation. Samples were centrifuged, and supernatant and cell pellet fractions were collected and stored separately at –80°C until used for analysis.

The vaginal supernatant was assayed for matrix metalloproteinase (MMP)-8, neutrophil gelatinase-associated lipocalin (NGAL), hyaluronan, extracellular MMP inducer (EMMPRIN) (R&D Systems, Minneapolis, MN), and alpha amylase (AbCam, Cambridge, United Kingdom) using commercial enzyme-linked immunosorbent assay kits. Concentrations of D- and L-lactic acid were determined colorimetrically using a commercial kit (BioAssay Systems, Hayward, CA). The total protein content of each vaginal supernatant was determined using a commercial colorimetric assay (Pierce, Rockford, IL). The lower limits of sensitivity were: 78 pg/mL for NGAL, 39 pg/mL for EMMPRIN, 62.5 pg/mL for MMP-8, 0.4 ng/mL for hyaluronan, 0.3 mU/mL for amylase, and 0.02 mmol/L for D- and L-lactic acid. Values of MMP-8, NGAL, hyaluronan, and EMMPRIN were converted to ng/μg or pg/μg total protein and mU/ug for amylase.

The cellular fraction was resuspended and lysed in a detergent-based buffer containing protease inhibitors, as described previously. The lysate was then centrifuged and the supernatant collected and immediately assayed for p62 protein and hsp70 using commercial enzyme-linked immunosorbent assay kits (R&D Systems, Minneapolis, MN/Enzo Life Sciences, Farmingdale, NY). p62 (also known as sequestsome-1) is a protein in the cytoplasm that binds to intracellular compounds that are destined to be engulfed by autophagosome and degraded. The p62 is simultaneously degraded and so the concentration of p62 that is detectable in the cytoplasm is inversely proportional to the extent of autophagy induction. The lower limit of sensitivity was 16 pg/mL for hsp70 and 100 pg/mL for p62. Laboratory personnel were blinded to all clinical data.

Statistical analysis was performed using GraphPad (Graphpad Software Inc, San Diego, CA) and SPSS v22 (IBM Corp, Armonk, NY). Categorical variables were evaluated using χ ² analysis or Fisher exact test for variables with low frequencies. Associations between the assays and GBS status were evaluated by the nonparametric Mann-Whitney U test since the values were not normally distributed. Correlations between the different mediators were examined by the Spearman rank correlation test. All P values were 2-sided, with alpha level of statistical significance set at .05.