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A Solution to Antifolate Resistance in Group B Streptococcus : Untargeted Metabolomics Identifies Human Milk Oligosaccharide-Induced Perturbations That Result in Potentiation of Trimethoprim

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Adjuvants can be used to potentiate the function of antibiotics whose efficacy has been reduced by acquired or intrinsic resistance. In the present study, we discovered that human milk oligosaccharides (HMOs) sensitize strains of group B Streptococcus (GBS) to trimethoprim (TMP), an antibiotic to which GBS is intrinsically resistant. Reductions in the MIC of TMP reached as high as 512-fold across a diverse panel of isolates. To better understand HMOs’ mechanism of action, we characterized the metabolic response of GBS to HMO treatment using ultrahigh-performance liquid chromatography–high-resolution tandem mass spectrometry (UPLC-HRMS/MS) analysis. These data showed that when challenged by HMOs, GBS undergoes significant perturbations in metabolic pathways related to the biosynthesis and incorporation of macromolecules involved in membrane construction. This study represents reports the metabolic characterization of a cell that is perturbed by HMOs. IMPORTANCE Group B Streptococcus is an important human pathogen that causes serious infections during pregnancy which can lead to chorioamnionitis, funisitis, premature rupture of gestational membranes, preterm birth, neonatal sepsis, and death. GBS is evolving antimicrobial resistance mechanisms, and the work presented in this paper provides evidence that prebiotics such as human milk oligosaccharides can act as adjuvants to restore the utility of antibiotics.
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A Solution to Antifolate Resistance in Group B Streptococcus:
Untargeted Metabolomics Identifies Human Milk
Oligosaccharide-Induced Perturbations That Result in
Potentiation of Trimethoprim
Schuyler A. Chambers,
a
Rebecca E. Moore,
a
Kelly M. Craft,
a
*Harrison C. Thomas,
a
Rishub Das,
a
Shannon D. Manning,
e
Simona G. Codreanu,
a,c
Stacy D. Sherrod,
a,c
David M. Aronoff,
b,f
John A. McLean,
a,c
Jennifer A. Gaddy,
b,d,f
Steven D. Townsend
a
a
Department of Chemistry, Vanderbilt University, Nashville, Tennessee, USA
b
Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, USA
c
Center for Innovative Technology, Nashville, Tennessee, USA
d
Department of Veterans Affairs, Tennessee Valley Healthcare Systems, Nashville, Tennessee, USA
e
Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, USA
f
Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA
Schuyler A. Chambers and Rebecca E. Moore contributed equally to this work. Author order was determined alphabetically.
ABSTRACT Adjuvants can be used to potentiate the function of antibiotics whose
efficacy has been reduced by acquired or intrinsic resistance. In the present study,
we discovered that human milk oligosaccharides (HMOs) sensitize strains of group B
Streptococcus (GBS) to trimethoprim (TMP), an antibiotic to which GBS is intrinsically
resistant. Reductions in the MIC of TMP reached as high as 512-fold across a diverse
panel of isolates. To better understand HMOs’ mechanism of action, we character-
ized the metabolic response of GBS to HMO treatment using ultrahigh-performance
liquid chromatography–high-resolution tandem mass spectrometry (UPLC-HRMS/MS)
analysis. These data showed that when challenged by HMOs, GBS undergoes signifi-
cant perturbations in metabolic pathways related to the biosynthesis and incorpora-
tion of macromolecules involved in membrane construction. This study represents
reports the metabolic characterization of a cell that is perturbed by HMOs.
IMPORTANCE Group B Streptococcus is an important human pathogen that causes
serious infections during pregnancy which can lead to chorioamnionitis, funisitis,
premature rupture of gestational membranes, preterm birth, neonatal sepsis, and
death. GBS is evolving antimicrobial resistance mechanisms, and the work presented
in this paper provides evidence that prebiotics such as human milk oligosaccharides
can act as adjuvants to restore the utility of antibiotics.
KEYWORDS group B Streptococcus, human milk oligosaccharides, resistance,
adjuvants, antifolate drugs
The development of antibiotics is arguably one of the most important advances in
modern medicine. Antibiotics can be organized according to the cellular compo-
nent or system they engage and whether they inhibit cell growth (bacteriostatic) or
induce cell death (bactericidal). Although antibiotics that target cellular viability are
effective, these agents impose selective pressures that foster the evolution of resistant
phenotypes (1). Combination therapy has emerged as a powerful solution to resistance
issues that plague monotherapy (2). This approach, which involves codosing an anti-
Citation Chambers SA, Moore RE, Craft KM,
Thomas HC, Das R, Manning SD, Codreanu SG,
Sherrod SD, Aronoff DM, McLean JA, Gaddy JA,
Townsend SD. 2020. A solution to antifolate
resistance in group B Streptococcus: untargeted
metabolomics identifies human milk
oligosaccharide-induced perturbations that
result in potentiation of trimethoprim. mBio
11:e00076-20. https://doi.org/10.1128/mBio
.00076-20.
Editor Jimmy D. Ballard, University of
Oklahoma Health Sciences Center
Copyright © 2020 Chambers et al. This is an
open-access article distributed under the terms
of the Creative Commons Attribution 4.0
International license.
Address correspondence to Jennifer A. Gaddy,
jennifer.a.gaddy@vanderbilt.edu, or Steven D.
Townsend, steven.d.townsend@vanderbilt.edu.
*Present address: Kelly M. Craft, Department of
Chemistry & Chemical Biology, Harvard
University, Cambridge, Massachusetts, USA.
This article is a direct contribution from David
M. Aronoff, a Fellow of the American Academy
of Microbiology, who arranged for and secured
reviews by Christian Melander, University of
Notre Dame; Amit Basu, Brown University; and
Xin Zhang, The Pennsylvania State University.
Received 15 January 2020
Accepted 30 January 2020
Published
RESEARCH ARTICLE
Therapeutics and Prevention
crossm
March/April 2020 Volume 11 Issue 2 e00076-20 ®mbio.asm.org 1
17 March 2020
biotic with an adjuvant that potentiates its function or a second antibiotic with a
different target, can improve efficacy and suppress resistance evolution (2–7).
One bacterial pathogen group that showcases multidrug resistance is group B
Streptococcus (GBS) (8). GBS is a leading cause of neonatal sepsis, pneumonia, and
meningitis (9–14). Recent data also suggest that GBS is a frequent cause of chorioam-
nionitis, endometritis, pneumonia, and urosepsis in adults with underlying medical
conditions (i.e., diabetes mellitus or immunosuppression) (15–19). As these patterns of
pathogenesis suggest, GBS is considered a saprophytic organism, i.e., invasive GBS
disease is most commonly observed in weakened hosts.
Treatment of GBS disease relies primarily on penicillin and ampicillin, followed by
first-generation cephalosporins and vancomycin (20). Alternative antibiotics, such as
lincosamides, are used for patients with
-lactam allergies. Due to resistance evolution,
macrolides, aminoglycosides, and tetracyclines are no longer clinically efficacious (21–
24). While our group and others have observed that GBS is resistant to a wide range of
antibiotics, GBS resistance remains poorly characterized and is a frontier of concern in
the clinic.
In the early stages of this program, we hypothesized that human milk oligosaccha-
rides (HMOs) possess antimicrobial and antivirulence properties (25). Indeed, we dis-
covered that heterogeneous HMOs modulate growth and biofilm production for a
number of bacterial pathogens (26, 27). We also determined the identities of several
single-entity HMOs with potent antimicrobial activity against GBS (28–31). In addition
to structure-activity relationship (SAR) studies, we found that HMOs potentiate the
activity of select intracellular targeting antibiotics (32). This included three antibiotics to
which GBS has evolved resistance, aminoglycosides, macrolides, and tetracyclines
(33–42). At their 50% inhibitory concentration (IC
50
), HMO extracts reduced the MICs of
certain intracellular targeting antibiotics up to 32-fold (Table 1). Interestingly, HMO
treatment did not affect
-lactam or glycopeptide activity; antibiotics that interfere
with cell wall synthesis. Based on this activity pattern, we hypothesized that HMOs
function by increasing membrane permeability, which would be an unprecedented
mode of action in GBS. This hypothesis was validated when HMOs were found to
increase membrane permeability by ca. 30% using a LIVE/DEAD BacLight assay (32).
Based on their ability to increase cellular permeability, a second-generation combi-
nation study was initiated to further characterize HMO enhancement of intracellular
targeting antibiotics in the GBS model. We took particular interest in trimethoprim
(TMP), an antifolate used in the treatment of enteric, respiratory, skin, and urinary tract
infections (43). Mechanistically, TMP is a bacteriostatic agent that inhibits dihydrofolate
reductase (DHFR), an enzyme within the folate biosynthesis pathway (44). Importantly,
interference with this pathway inhibits pyrimidine and purine biosynthesis, with down-
stream effects on bacterial DNA synthesis. Furthermore, a wide range of streptococcal
strains, including GBS, are intrinsically resistant to TMP (45–51). Resistance is typically
mediated by one of the following five mechanisms: (i) poor membrane permeability, (ii)
an impervious DHFR, (iii) mutations in the inherent DHFR, (iv) upregulation of gene
expression or gene duplication to increase DHFR production, and (v) horizontal transfer
of dfr genes that encode resistant DHFRs (45). We hypothesized that if TMP has
difficulty gaining penetrance into the GBS cell, HMOs could be used to sensitize GBS to
TABLE 1 Established patterns of HMO potentiation of antibiotic activity
Antibiotic in THB medium (strain)
MIC (
g/ml)
Fold reductionOverall With 5.0 mg/ml HMOs
Penicillin (CNCTC 10/84) 0.03 0.015 2
Vancomycin (CNCTC 10/84) 2 1 2
Clindamycin (GB2) 0.0312 0.0078 4
Gentamicin (GB590) 16 1 16
Erythromycin (GB590) 0.0312 0.001 32
Minocycline (CNCTC 10/84) 0.0625 0.0019 32
Chambers et al. ®
March/April 2020 Volume 11 Issue 2 e00076-20 mbio.asm.org 2
TMP. Described herein are the results of testing this hypothesis using heterogeneous
HMO extracts. To further evaluate the mechanism of HMO sensitization, ultrahigh-
performance liquid chromatography–high-resolution tandem mass spectrometry anal-
ysis (UPLC-HRMS/MS) was used to characterize the immediate metabolic response of
GBS to HMO-induced perturbations.
RESULTS AND DISCUSSION
HMOs demonstrate synergy with TMP against group B Streptococcus.HMOs
were isolated from donor breast milk and pooled to create two HMO cocktails; the first
(HMO-1) used milk from 10 donors, while the second (HMO-2) used milk from 7 donors.
Prior to potentiation studies, the MIC of the HMOs and TMP were determined sepa-
rately in each strain of GBS grown in Todd-Hewitt broth (THB) using a broth microdi-
lution assay (Table 2). HMOs were assayed against five strains of GBS of various
serotypes to determine the strain specificity of antibiotic potentiation. The strains
selected are all clinical isolates. CNCTC 10/84 is commercially available (52). Isolates
GB00590, GB00002, GB00651, and GB00083 were recovered from colonized pregnant
women (53, 54). GBS strains are divided into 10 serotypes (1a, 1b, and II to IX) based on
a serological reaction against their capsular polysaccharides (55). GB2, GB590, and
CNCTC 10/84 are serotypes Ia, III, and V, respectively. These three serotypes are the
most common isolates associated with early-onset disease in the United States and
together account for over 80% of all isolates (56). GB651 and GB83 are serotypes Ib and
IV, respectively. Globally, the five strains represent 85% of all isolate serotypes (57).
HMOs were dosed at their 25% inhibitory concentrations (IC
25
s) in CNCTC 10/84 and
GB2. The growth of the remaining GBS strains were so rapidly affected by HMO
treatment that subsequent IC
50
curve fitting yielded immeasurable confidence limits.
For these strains, the IC
25
from a similar strain having a superior fit dose-response curve
was used (see Fig. S1A to E in the supplemental material). In each strain, the MIC of TMP
was 512
g/ml or higher (Table 2). In GB2, a 512-fold reduction in MIC was observed.
A 256-fold reduction in MIC was observed in CNCTC 10/84. For the three remaining
strains (GB651, GB83, and GB590), the fold reductions in MIC were 16, 16, and 64,
respectively. The potentiation patterns described above are remarkable for several
reasons. First, they represent the greatest magnitude of antibiotic enhancement that
we have observed. Second, GBS is not susceptible to antifolate antibiotics, so the
chemotherapeutic regime is effective at sensitizing GBS to TMP.
Next, checkerboard assays were conducted with GB2 and GB590, stains for which
strong and weak potentiation of TMP were observed, respectively, to determine if the
HMO-TMP combination was synergistic or additive in nature (Fig. S2A and B). Synergy
is measured using the fractional inhibitory concentration (FIC) index value and is
defined when the FIC is 0.5 for each combination of compounds. It was demonstrated
that in GB590, synergy was achieved when dosing HMOs from 1.28 to 2.56 mg/ml in
combination with TMP dosed at 8 to 128
g/ml (FIC values, 0.281 to 0.508). In GB2,
the combination was synergistic with treatment of HMOs between 0.64 and 1.28 mg/ml
in conjunction with TMP at 4 to 32
g/ml (FIC values, 0.281 to 0.508). These assays
firmly demonstrate the HMO-TMP combination to be synergistic, and they characterize
the dosing windows required to achieve this effect.
TABLE 2 HMO potentiation of TMP
Strain in THB medium
MIC (
g/ml) for:
Fold reductionHMOs TMP
TMP with
1.42 mg/ml HMO
CNCTC 10/84 5.12
a
1,024 8
a
256
GB2 2.56
a
1,024 2
a
512
GB590 5.12
a
1,024 32
a
64
GB651 5.12
b
512 32
b
16
GB83 5.12
b
1,024 128
b
16
a
HMO-1.
b
HMO-2.
HMO Changes Enhance Antibiotics against GBS ®
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After evaluating the level of synergy in the cocktail, we conducted an experiment to
validate whether growth inhibition was due to HMO enablement of cognate engage-
ment of TMP with the folate pathway. By inhibiting folate biosynthesis, antifolates
inhibit the de novo biosynthesis of thymidylate and purine nucleotides. However, in
addition to de novo synthesis, cells can produce these nucleotides via salvage pathways
that use free thymidine or purine bases as precursors for the corresponding nucleo-
tides. Thus, we hypothesized that if HMOs facilitate TMP inhibition of the de novo
synthesis pathway, the addition of the preformed nucleotide precursors thymidine or
hypoxanthine would dampen the growth inhibitory capabilities of the HMO-TMP
combination, as thymidine and hypoxanthine serve as precursors in the pyrimidine and
purine salvage pathways, respectively (58).
In the experiment, we evaluated the MIC of the HMO-TMP cocktail in the presence
of thymidine (Table 3). The experiment was conducted in strains GB2 and GB590.
Against GB2, HMO supplementation decreased the MIC of TMP from 1,024
g/ml to
2
g/ml (512-fold reduction). Against GB590, the MIC of TMP was reduced from
1,024
g/ml to 32
g/ml (at least a 64-fold reduction) (Table 2). In the presence of
added thymidine, the MICs of TMP in the HMO-TMP combination increased 8-fold to
16
g/ml in GB2 and 4-fold to 128
g/ml in GB590. These results support the hypoth-
esis that supplemental thymidine mitigates the effects of the HMO-TMP combination
and is able to partially salvage the folate biosynthetic pathway. Importantly, the MIC of
the HMO cocktail individually did not change in the presence of added thymidine. This
indicates that the folate pathway is not a target for HMOs. We therefore conclude that,
in the presence of HMOs, TMP gains penetrance into the group B streptococcal cell and
exhibits on-target inhibition of the folate cycle.
The final assay in this study was a comparison of the HMO-TMP combination with
the clinically useful TMP-sulfadiazine (SDZ) combination (Table S2). In both GB590 and
GB2, the TMP-sulfadiazine combination was largely ineffective, with an MIC of 512
g/ml, while the HMO-TMP combination sees the potentiation profile described above
(Table 2). This result demonstrates that while antifolate-based antibiotic combination
treatments remain largely ineffective against GBS, the HMO-TMP combination is oper-
ative. This insight offers new consideration for the use of existing combination thera-
pies in patient care.
Characterizing the HMO mode of action using untargeted metabolomics. The
mode of action of an antimicrobial agent cannot accurately be described in terms of a
single static target; rather, the complete induced response must be evaluated. In
theory, a single chemotherapeutic could have a wide range of direct and indirect
targets, simultaneously interfering with multiple enzymes or pathways. Accordingly,
the final stage of the study focused on utilizing global, untargeted metabolomic
analysis to characterize the early response of GBS to HMO-mediated perturbations. The
analysis described provides HMO-mediated perturbations. For this study, strain GB2
was used, as it was most susceptible to treatment with HMOs (MIC, 2.56
g/ml). Two
groups were analyzed and compared, with the first being an untreated GB2 control and
the second being GB2 treated with HMOs dosed at 1 mg/ml. This concentration
promoted cellular death (ca. 20 to 40%) compared to the untreated controls but also
provided enough remaining cellular mass for analysis (minimum, 200
g).
Our experimental design from sample collection through data analysis is depicted in
Fig. 1A. The annotated and statistically significant metabolites observed in the exper-
iment (Fig. 1B) were subjected to traditional pathway analysis (Fig. S3). The results
TABLE 3 HMO potentiation of TMP in the presence of thymidine
Strain in THB medium plus
20
g/ml thymidine
MIC (
g/ml) for:
Fold reductionTMP
TMP with HMO-1
(dose [mg/ml])
GB2 1,024 16 (1.43) 64
GB590 1,024 128 (1.42) 8
Chambers et al. ®
March/April 2020 Volume 11 Issue 2 e00076-20 mbio.asm.org 4
showed the most statistically perturbed metabolic pathways to be linoleic acid metab-
olism, sphingolipid metabolism, glycerophospholipid metabolism, and pyrimidine me-
tabolism (Fig. 1C and D). Characterized below are perturbations to both linoleic acid
and glycerophospholipid metabolism (Fig. 2 and 3). We focus on these pathways, as
each is critical to membrane formation and structural integrity, i.e., each pathway
contributes to the synthesis of membrane-bound macromolecules and their corre-
sponding precursors (59).
Based on statistical significance, linoleic acid metabolism is the metabolic pathway
most impacted when GBS is exposed to HMOs (Fig. 2A and S4). Linoleic acid meta-
bolites play a critical role in both cellular signaling and the stress response. Each is also
critical to proper membrane construction (60, 61). All identified linoleic acid metabolites
were accumulated in the HMO-treated population, with several metabolites having a
100-fold increase from the untreated controls (Fig. 2B). Two epoxyoctadecanoic acid
metabolites were of particular interest, epoxyoctadecanoic acids (EpOMEs) and dihy-
droxyoctadecanoic acids (DiHOMEs). Accumulation of these metabolites is linked to
changes in Na
and K
ion channels and, subsequently, cell membrane fluidity (62). In
addition to the roles of EpOMEs and DiHOMEs in cell membrane construction, linoleic
acid metabolites have been shown to have a critical role in cellular signaling and the
stress response.
Glycerophospholipid metabolism was also significantly impacted, and in general, we
observed an accumulation of these metabolites compared to the control (Fig. 3A and
S5). Glycerophospholipid metabolites were observed with significant fold changes
compared to the control. For example, PE(17:0/0:0), PE(P-16:0/0:0), and PE(19:1/12:0),
known degradation products of phosphatidylethanolamine (PE), one of three major
components of the cellular membrane, were observed to have up to a 50-fold change
FIG 1 Workflow and pathway analysis using global, untargeted metabolomics data analysis. (A) Overview of global,
untargeted metabolomic workflow. (B) Global output of identified metabolites from RPLC and HILIC methods and
subsequent filtering for significance according to a Pvalue of 0.05 and fold change of |2|. (C) Table output of metabolic
pathway enrichment analysis. The number of total metabolites in the pathway, the number of hits, and the Pvalue were
calculated using MetaboAnalyst 4.0. CoA, coenzyme A. (D) Metabolomic pathway analysis visualization. Shown is a
graphical representation analysis using the statistically significant metabolite compounds (P0.05; fold change, |2|)
annotated from RPLC and HILIC analyses. Matched pathways were arranged by Pvalues (from pathway enrichment
analysis) on the yaxis, and pathway impact values (from pathway topology analysis) are shown on the xaxis; node color
is based on pathway Pvalue, and node radius is determined based on pathway impact values; individual nodes represent
individual pathways.
HMO Changes Enhance Antibiotics against GBS ®
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increase compared to the untreated control (63). This observed accumulation of lipid
metabolites indicates an increased rate of breakdown of critical cell membrane com-
ponents when bacteria are dosed with HMOs. In fact, this type of metabolite accumu-
lation has been previously observed in other model organisms when exposed to
antibiotics (64–67).
In addition to dysregulating pathways directly related to the production of mem-
brane components, HMOs perturbed additional pathways related to essential cellular
function. These include, for example, increased accumulation of purine and pyrimidine
nucleotide precursors. An inability to synthesize nucleotides would lead to perturba-
tions in DNA and RNA synthesis. HMO treatment also led to an accumulation of
metabolites in the cysteine and methionine metabolic pathway. Cysteine biosynthesis
is the primary pathway for incorporating sulfur into cellular components. In addition to
serving as a precursor of methionine, cysteine is also the direct precursor to biotin,
thiamine, and lipoic acid. Methionine is an essential amino acid in all organisms, as it
is both proteinogenic and a component of the cofactor S-adenosyl methionine. Inter-
estingly, sphingolipid metabolism was observed to be significantly perturbed upon
HMO treatment. While GBS does not synthesize sphingolipids directly, it does rely on
the host for access to these compounds for the biosynthesis of cell membrane
components. This metabolic change could suggest that HMO treatment has an impact
on host-microbe interactions (68, 69). Finally, several cell wall synthesis-associated
metabolites were also identified as being accumulated in the treated sample, but a
higher experimental mass range would be needed to identify a more significant
FIG 2 Linoleic acid-associated metabolite identification and statistical representation. (A) Heat map visualization of the
significantly differently regulated linoleic acid metabolic pathway upon HMO treatment. Linoleic acid metabolism
members shown here were detected by RPLC-positive LC-MS/MS analysis. Samples (columns) and metabolite compounds
(rows) were processed using Euclidean average clustering via MetaboAnalyst 4.0. The heat map was generated for
Pareto-scaled, log-transformed data, and colors are displayed by relative abundance, ranging from low (blue) to high (red),
as shown in the legend. (B) Corresponding data table of linoleic acid metabolites, where the asterisk (*) denotes
significance with a Pvalue of 0.05 and fold change of |2|. ODE, octadecadienoic acid; 13-HOTE, 13-hydroxyoctadeca-
9,11,15-trienoic acid; 9,10-DiHOME, 9,10-dihydroxyoctadec-12-enoic acid; 9,12,13-TriHOME, 9,12,13-trihydroxyoctadecanoic
acid; 13S-HODE, 13S-octadecadienoic acid; 9(10)-EpHOME, 9(10)-epoxyhydroxyoctadecanoic acid; ID, identifier.
Chambers et al. ®
March/April 2020 Volume 11 Issue 2 e00076-20 mbio.asm.org 6
amount of these precursors and better identify a global trend. The variety of metabolic
pathways perturbed not only illuminates the synergistic nature of the TMP and HMO
combination treatment but demonstrates that antimicrobial agents have broad effects
on cellular biology (Fig. S6).
In summary, this study demonstrates that HMOs potentiate trimethoprim in group
BStreptococcus bacteria, with a synergistic profile that spans the most prevalent
serotypes worldwide. This potentiation profile makes antifolate antibiotics of potential
use in an organism where they have been long considered resistant. Moreover, this
combination could represent an alternative treatment for GBS-positive mothers with
penicillin allergies given the high rates of resistance observed with alternative agents.
To characterize the mechanism of HMO-mediated antimicrobial activity, we have
presented the first global, untargeted metabolomic analysis of HMO-mediated pertur-
bations within any cell type and have shown significant impacts on cell membrane-
affiliated macromolecules. While a number of high-throughput methods have been
performed to elucidate an antibiotic’s mode of action (e.g., cytological profiling, genetic
screens, or gene expression and proteomic profiling), direct experimental evidence that
FIG 3 Glycerophospholipid-associated metabolite identification and statistical representation. (A) Heat map visualization
of the significantly differently regulated glycerophospholipid metabolism pathway upon HMO treatment. Glycerophos-
pholipid members shown here were detected by HILIC-positive LC-MS/MS analysis. Samples (columns) and metabolite
compounds (rows) were processed using Euclidean average clustering via MetaboAnalyst 4.0. The heat map was generated
for Pareto-scaled, log-transformed data, and colors are displayed by relative abundance, ranging from low (blue) to high
(red), as shown in the legend. SM, sphingomyelin; PC, phosphocholine; PI, phosphoinositol; DG, diglyceride; LysoPC,
lysophosphatidylcholine. (B) Corresponding data table of glycerophospholipid metabolites, where the asterisk (*) denotes
significance with a Pvalue of 0.05 and fold change of |2|.
HMO Changes Enhance Antibiotics against GBS ®
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rapid metabolic changes are causal in facilitating the microbial response to antibiotics
is lacking. In fact, little is described about the downstream phenotypic changes induced
by antimicrobial agents. In the future, metabolomic experiments will be employed to
better describe the phenotypic response of GBS to HMO-induced effects. We hypoth-
esize that metabolomics will enable the characterization of the indirect connections
critical to HMOs’ mechanism of action. Since metabolites present the final phenotypic
manifestation of an organism and the final endpoint of biochemical reactions reflects
the interplay between gene expression, protein function, and the environment, we
argue that further metabolomics analyses are necessary to understand the HMO mode
of action (70, 71).
MATERIALS AND METHODS
Antibiotics and additional chemicals. Trimethoprim lactate 98% was purchased from Alfa Aesar.
-Galactosidase from Kluyveromyces lactis, at 2,600 units/g, was purchased from Sigma-Aldrich. Aceto-
nitrile (ACN; catalog no. A955-1), methanol (MeOH; catalog no. A456-1), and water (catalog no. W6-1,
liquid chromatography-mass spectrometry [LC-MS] grade; Optima) for the mass spectrometry analysis
were obtained from Thermo Fisher Scientific.
HMO isolation. Human milk was obtained from 17 healthy, lactating women between 3 days and
3 months postpartum and stored between 80 and –20°C. Deidentified milk was provided by Jörn-
Hendrik Weitkamp from the Vanderbilt Department of Pediatrics, under a collection protocol approved
by the Vanderbilt University institutional review board (IRB no. 100897), or from Medolac. Milk samples
were thawed and then centrifuged for 45 min. Following centrifugation, the resultant top lipid layer was
removed. The proteins were then removed by diluting the remaining sample with roughly 1:1 (vol/vol)
180 or 200 proof ethanol, chilling the sample briefly, and centrifuging for 45 min, followed by removal
of the resulting HMO-containing supernatant. Following concentration of the supernatant in vacuo, the
HMO-containing extract was dissolved in 0.2 M phosphate buffer (pH 6.5) and heated to 37°C.
-Galactosidase from Kluyveromyces lactis was added, and the reaction mixture was stirred until lactose
hydrolysis was complete. The reaction mixture was diluted with roughly 1:0.5 (vol/vol) 180 or 200 proof
ethanol, chilled briefly, and then centrifuged for 30 min. The supernatant was removed and concentrated
in vacuo, and the remaining salts, glucose, and galactose were separated from the oligosaccharides using
size exclusion chromatography with P-2 gel (H
2
O eluent). The oligosaccharides were then dried by
lyophilization. Correspondingly, HMO isolates from donors were combined and solubilized in water to
reach a final concentration of 102.6 mg/ml.
Bacterial strains and culture conditions. The bacterial strains are shown in Table S1. All strains were
grown on tryptic soy agar plates supplemented with 5% sheep blood (blood agar plates) at 37°C in
ambient air overnight. All strains were subcultured from blood agar plates into 5 ml of Todd-Hewitt broth
(THB) and incubated under shaking conditions at 180 rpm at 37°C overnight. Following overnight
incubation, bacterial density was quantified through absorbance readings at 600 nm (OD
600
) using a
Promega GloMax-Multi detection system plate reader. Bacterial numbers were determined using the
predetermined coefficient of an OD
600
of 1, equal to 10
9
CFU/ml.
Broth microdilution method for determination of MICs. All strains were grown overnight as
described above and used to inoculate fresh THB or THB plus 20
g/ml thymidine to achieve 5 10
5
CFU/ml. To 96-well tissue culture-treated, sterile polystyrene plates was added the inoculated medium
in the presence of increasing concentrations of antibiotic or HMO cocktail to achieve a final volume of
100
l per well. Bacteria grown in medium in the absence of any compounds served as the controls. The
plates were incubated under static conditions at 37°C in ambient air for 24 h. Bacterial growth was
quantified through absorbance readings (OD
600
). The MICs were assigned at the lowest concentration of
compound at which no bacterial growth was observed.
Broth microdilution method for antibiotic combination. All strains were grown overnight as
described above and the subcultures used to inoculate fresh THB or THB plus 20
g/ml thymidine to
achieve 5 10
5
CFU/ml. Freshly inoculated medium was then supplemented with HMOs at their IC
25
.To
96-well tissue culture-treated, sterile polystyrene plates was added the inoculated medium supple-
mented with HMOs in the presence of increasing concentrations of antibiotic. Bacteria grown in medium
in the absence of any compounds served as one control. Bacteria grown in medium supplemented with
HMOs in the absence of any antibiotic served as a second control. MICs were determined as previously
described.
Synergy assay. Group B Streptococcus strains (GB2 and GB590) were grown overnight as described
above and used to inoculate fresh THB to achieve 5 10
5
CFU/ml. One hundred microliters per well of
inoculated medium was added to 96-well tissue culture-treated, sterile polystyrene plates. Trimethoprim
was 2-fold serially diluted descending down the plate to achieve a final volume of 100
l per well. The
final row was left without any trimethoprim. The HMO cocktail was 2-fold serially diluted going from right
to left across the plate. The far-left column was left without any HMO cocktail. Bacteria grown in medium
in the absence of either compound served as the controls. The plates were incubated under static
conditions at 37°C in ambient air for 24 h. Bacterial growth was quantified through absorbance readings
(OD
600
). The MICs were assigned at the lowest concentration of compound at which no bacterial growth
was observed. The fractional inhibitory concentration (FIC) index was used to evaluate synergy. The
calculation of the FIC index is as follows: FIC FIC A FIC B (MIC of drug A in the combination/MIC
of drug A alone) (MIC of drug B in the combination/MIC of drug B alone), where A is trimethoprim and
Chambers et al. ®
March/April 2020 Volume 11 Issue 2 e00076-20 mbio.asm.org 8
B is the HMO cocktail. The combination is considered synergistic when the FIC is 0.5, additive or
indifferent when the FIC is 0.5 to 4, and antagonistic when the FIC is 4.
Statistical analysis. The data for the HMO antimicrobial and combination assays represent 3
biological replicates, each with 3 technical replicates. The data for the synergy assays represent 3
biological replicates. Data are expressed as the mean biomass standard error of the mean (SEM).
Statistical analyses were performed in the GraphPad Prism software v. 7.0c. Statistical significance was
determined using one-way analysis of variance (ANOVA) with post hoc Dunnett’s multiple-comparison
test comparing growth in the presence of ca. 5 mg/ml HMOs to growth in medium alone. HMO IC
50
curves were generated in the GraphPad Prism software v. 7.0c using an inhibition dose-response
nonlinear regression curve fit for log(inhibitor) versus normalized response with a variable slope.
Sample preparation for metabolomic analysis. Group B Streptococcus strain GB2 was grown
overnight as described above and used to inoculate 10 ml of fresh THB medium to achieve 5 10
5
CFU/ml. Untreated GB2 in 10 ml of medium served as a control, while other GB2 cultures were treated
with HMOs at 1.00 mg/ml. After 24 h, the samples were centrifuged at 1,500 rpm for 20 min to generate
a bacterial pellet. The medium was removed and the pellet washed with 200
l of 50 mM ammonium
formate buffer. The pellet was then resuspended in 200
l of 50 mM ammonium formate buffer and
transferred to a sterile Eppendorf tube. This was then centrifuged at 1,500 rpm for 10 min to generate
a bacterial pellet. The buffer was removed and the pellet flash frozen in liquid N
2
and stored until use.
The bacterial cell pellets were lysed using 400
l ice-cold lysis buffer (1:1:2, AcCN:MeOH:ammonium
bicarbonate 0.1 M [pH 8.0], LC-MS grade) and vortexed. Individual samples were sonicated using a probe
tip sonicator, with 10 pulses at 30% power and cooling down in ice between samples. A bicinchoninic
acid (BCA) protein assay was used to determine the protein concentration for each individual sample and
adjusted to a total amount of protein of 200
gin200
l of lysis buffer. Isotopically labeled standard
molecules phenylalanine-D8 (CDN Isotopes, Quebec, CA) and biotin-D2 (CIL, MA, USA) were added to
each sample to assess sample preparation reproducibility. Metabolites were extracted from untreated
control and HMO-treated cultures using protein precipitation by the addition of 800
l of ice-cold
methanol (4by volume) and incubated overnight at –80°C. Following incubation, samples were
centrifuged at 10,000 rpm for 10 min to eliminate precipitated proteins, and the metabolite-containing
supernatant was dried in vacuo and stored at –80°C until further UPLC-HRMS/MS analysis.
Global untargeted metabolomic analyses. Metabolite extracts were analyzed using reverse-phase
liquid chromatography (RPLC) and hydrophilic interaction liquid chromatography (HILIC), followed by
subsequent mass spectrometry analysis using a high-resolution Q-Exactive high-fidelity (HF) hybrid
quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with a
Vanquish ultrahigh-performance liquid chromatography (UHPLC) binary system and autosampler
(Thermo Fisher Scientific, Bremen, Germany). A quality control sample was prepared by pooling equal
volumes of each sample. Isotopically labeled standards tryptophan-D3, carnitine-D9 (CDN Isotopes,
Quebec, CA), valine-D8, and inosine-4N15 (CIL, MA, USA) were added to each sample to assess MS
instrument reproducibility.
Metabolite extracts (10-
l injection volume) were separated on a SeQuant ZIC-HILIC 3.5-
m, 2.1-
mm by 100-mm column (Millipore Corporation, Darmstadt, Germany) held at 40°C for the HILIC analysis.
Liquid chromatography was performed at 200
l/min using solvent A (5 mM ammonium formate in 90%
water, 10% acetonitrile) and solvent B (5 mM ammonium formate in 90% acetonitrile, 10% water) with
the following gradient: 95% B for 2 min, 95 to 40% B over 16min, 40% B held for 2 min, and 40 to 95%
B over 15 min, and 95% B held for 10 min (gradient length, 45 min). For the RPLC analysis, metabolite
extracts (10
l injection volume) were separated on a Hypersil Gold, 1.9
m, 2.1-mm by 100-mm column
(Thermo Fisher) held at 40°C. Liquid chromatography was performed at 250
l/min using solvent A (0.1%
formic acid [FA] in water) and solvent B (0.1% FA in acetonitrile [ACN]) with the following gradient: 5%
B for 1 min, 5 to 50% B over 9 min, 50 to 70% B over 5 min, 70 to 95% B over 5 min, 95% B held for 2 min,
95 to 5% B over 3 min, and 5% B held for 5 min (gradient length, 30 min).
MS analyses were acquired over a mass range of m/z 70 to 1,050 using electrospray ionization
positive mode. MS scans were analyzed at a resolution of 120,000, with a scan rate of 3.5 Hz. The
automatic gain control (AGC) target was set to 1 10
6
ions, and the maximum injection time (IT) was at
100 ms. Source ionization parameters were optimized, and these include spray voltage, 3.0 kV; transfer
temperature, 280°C; S-lens level, 40; heater temperature, 325°C; sheath gas, 40; aux gas, 10; and sweep
gas flow, 1. Tandem spectra were acquired using a data-dependent acquisition (DDA) in which one MS
scan is followed by 2, 4, or 6 tandem MS (MS/MS) scans. MS/MS scans are acquired using an isolation
width of m/z 1.3, stepped normalized collision energy (NCE) of 20 and 40, and a dynamic exclusion for
6 s. MS/MS spectra were collected at a resolution of 15,000, with an AGC target set at 2 10
5
ions and
maximum IT of 100 ms. Instrument performance and reproducibility in the run sequence were assessed
by monitoring the retention times and peak areas for the heavy labeled standards added to the
individual samples prior to and after metabolite extraction to assess sample processing steps and
instrument variability (Table S3).
Metabolomics data processing. UPLC-HRMS/MS raw data were imported, processed, normalized,
and reviewed using Progenesis QI v.2.1 (Nonlinear Dynamics, Newcastle, UK). All MS and MS/MS sample
runs were aligned against a quality control (pooled) reference run, and peak picking was performed on
individual aligned runs to create an aggregate data set. Following peak picking, unique spectral features
(retention time and m/z pairs) were grouped based on adducts and isotopes, and individual features or
metabolites were normalized to all features. Compounds with 25% coefficient of variance (CV) were
retained for further analysis. Pvalues were calculated by Progenesis QI using variance-stabilized
HMO Changes Enhance Antibiotics against GBS ®
March/April 2020 Volume 11 Issue 2 e00076-20 mbio.asm.org 9
measurements achieved through log normalization, and metabolites with a Pvalue of 0.05 calculated
by one-way analysis of variance (ANOVA) and with a fold change (FC) of |2| were considered significant.
Tentative and putative identifications were performed within Progenesis QI using accurate mass
measurements (5 ppm error), isotope distribution similarity, and fragmentation spectrum matching
based on database searches against the Human Metabolome Database (HMDB), METLIN and National
Institute of Standards and Technology (NIST) databases, and an in-house database (72–76). Annotations
from both RPLC and HILIC analyses were performed for all significant compounds (P0.05, FC |2|).
Annotations were further analyzed using pathway overrepresentation analysis using MetaboAnalyst 4.0
(77, 78). The level system for metabolite identification confidence was used. The level 3 (L3) of confidence
for the metabolite identifications was assigned for those molecules that showed minimal experimental
evidence compared to level 2 (L2) but do prioritize a top candidate. These are accepted by the
metabolomics community and represent families of molecules that cannot be distinguished by the data
acquired, predominantly because there are too many isomers as possible candidate metabolites, but the
family trends can be informative as well.
SUPPLEMENTAL MATERIAL
Supplemental material is available online only.
FIG S1, DOCX file, 0.1 MB.
FIG S2, DOCX file, 0.1 MB.
FIG S3, DOCX file, 0.1 MB.
FIG S4, DOCX file, 0.2 MB.
FIG S5, DOCX file, 0.4 MB.
FIG S6, DOCX file, 0.1 MB.
TABLE S1, DOCX file, 0.1 MB.
TABLE S2, DOCX file, 0.1 MB.
TABLE S3, DOCX file, 1.1 MB.
ACKNOWLEDGMENTS
This work was supported by the National Science Foundation (CAREER award to
S.D.T., CHE-1847804). S.D.T. is supported by a Dean’s Faculty Fellowship from the
College of Arts & Science at Vanderbilt University. J.A.G., D.M.A., and S.D.M. acknowl-
edge support from the NIH under grants R01-HD090061, R01-AI134036, and U01-
TR02398, and from the March of Dimes Foundation. H.C.T. was supported by an
undergraduate research fellowship from the Fleischer family.
The donor mothers are acknowledged for their generous contributions.
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Chambers et al. ®
March/April 2020 Volume 11 Issue 2 e00076-20 mbio.asm.org 12
... Moreover, HMOs may protect against neonatal pathogens by acting as soluble "decoy" receptors for enteric pathogens (17,18), through neutralization of bacterial toxins (19,20), or via direct antimicrobial activity, including against GBS (21)(22)(23)(24). Although the mechanism of HMO-mediated GBS inhibition is not known, GBS expression of a putative glycosyltransferase (locus san_0913) is necessary for inhibitory activity (21), and HMO exposure lowers GBS sensitivity to antibiotics, including vancomycin, erythromycin, and trimethoprim (21,25,26). Additional support for HMO-mediated anti-GBS activity stems from clinical observations that mothers who produce a functional variant of the fucosyltransferase enzyme FUT3, which attaches fucose in an a1-3 or a1-4 linkage to form certain HMOs, are less likely to be vaginally colonized by GBS (27). ...
... HMO resistance conferred by disruption of san_0913 does not alter GBS biofilm formation, adherence, susceptibility to antibiotics, or in vivo colonization in the absence of HMOs. Although the exact mechanism of HMO anti-GBS activity has yet to be established, increased GBS sensitivity to intracellular targeting antibiotics and enhanced cell membrane permeability occur following HMO exposure (21,25,26). In addition, HMO exposure perturbs multiple GBS metabolic pathways including those related to linoleic acid, sphingolipid, glycerophospholipid, and pyrimidine metabolism (26). ...
... Although the exact mechanism of HMO anti-GBS activity has yet to be established, increased GBS sensitivity to intracellular targeting antibiotics and enhanced cell membrane permeability occur following HMO exposure (21,25,26). In addition, HMO exposure perturbs multiple GBS metabolic pathways including those related to linoleic acid, sphingolipid, glycerophospholipid, and pyrimidine metabolism (26). A transposon mutant library screen identified the gbs0738 gene (locus san_0913 or GBSCOH1_RS04065 in COH1), a putative glycosyltransferase family 8 protein, as essential for GBS susceptibility to HMOs over a 7-h time course (21); however, the functional role of this glycosyltransferase in GBS-host interactions and resistance to antimicrobial compounds has not been characterized. ...
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During pregnancy, GBS ascension into the uterus can cause fetal infection or preterm birth. In addition, GBS exposure during labor creates a risk of serious disease in the vulnerable newborn and mother postpartum.
... Specific human milk oligosaccharides (HMOs) were able to increase the permeability of the membrane of a Gram-positive coccus, group B Streptococcus (GBS), in a concentration-dependent manner to exert anti-bacterial activity [25]. Moreover, increased membrane permeability also contributed to the improvement of antibiotic efficacy, including gentamicin (aminoglycosides), erythromycin (macrolides) and minocycline (tetracyclines) [26]. HMOs can also increase the efficacy of aminoglycosides (but not of β-lactams) against Acinetobacter baumannii, a Gram-negative bacterium [27]. ...
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Emerging antimicrobial resistance in infections asks for novel intervention strategies. Galacto-oligosaccharides (GOS) might be attractive alternatives to antibiotics due to their anti-inflammatory and anti-adhesive properties. Mannheimia haemolytica is one of the major Pasteurellaceae associated with bovine lung infections. Using M. haemolytica, we demonstrated that GOS have the capacity to reduce bacterial viability and can be used as adjuvant to improve antibiotic efficacy. Using M. haemolytica-treated primary bronchial epithelial cells (PBECs) of calves, we identified the anti-adhesive and anti-invasive activities of GOS. The observed inhibition of cytokine/chemokine release and the prevention of airway epithelial barrier dysfunction in M. haemolytica-treated PBECs by GOS might be related to the downregulation of “toll-like receptor 4/nuclear factor-κB” pathway and the anti-invasive and anti-adhesive properties of GOS. Particularly, GOS lowered lipopolysaccharides- but not flagellin-induced cytokine/chemokine release in calf and human airway epithelial cells. Finally, we performed in vivo experiments with calves and demonstrated for the first time that intranasal application of GOS can relieve lung infections/inflammation and lower M. haemolytica positivity in the lungs without affecting clinical performance. These findings not only shed light on the anti-inflammatory mechanisms of GOS during lung infections, but GOS might also be a promising anti-bacterial agent for preventing (lung) infections.
... Such HMO fragilized bacteria were shown to be more sensitive to antibiotic treatment (78; 81; 153) . Interestingly, Chambers et al. reported increased 12,13-DiHOME production in GBS treated with HMOs (81) . Possibly, in vivo this may trigger an increased effector immune response for pathogen clearance. ...
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Human milk oligosaccharides (HMOs) have been researched by scientists for over 100 years, driven by the substantial evidence for the nutritional and health benefits of mother's milk. Yet research has truly bloomed during the last decade, thanks to the progress in biotechnology, which allowed the production of large amounts of bona fide HMOs. The availability of HMOs has been particularly crucial for the renewed interest in HMO research because of the low abundance or even absence of HMOs in farmed animal milk. This interest is reflected in the increasing number of original research publications and reviews on HMOs. Here, we provide an overview and critical discussion on structure function relations of HMOs that highlight why they are such interesting and important components of human milk. Clinical observations in breastfed infants backed by basic research from animal models provide guidance as to what physiological roles for HMOs are to be expected. From an evidence-based nutrition viewpoint, we discuss the current data supporting clinical relevance of specific HMOs based on randomized placebo controlled clinical intervention trials in formula-fed infants. This article is protected by copyright. All rights reserved.
... Structurally composed of five monosaccharide units, HMOs have antibacterial activity against both gram-positive and gram-negative pathogens 19 . We have previously demonstrated the bactericidal power and anti-biofilm activity of HMOs in a variety of bacterial pathogens 20,21 . While our previous work indicated HMOs did not have potent bactericidal activities against a single laboratory strain of A. baumannii, here we present our further investigations into the antibiofilm properties of these molecules, which have broad activity against a range of clinical strains of A. baumannii 19 . ...
... Individual samples were spun down to remove proteins and the subsequent supernatant was used for analyses. Samples were separated and analyzed using reverse-phase liquid chromatography connected to a Thermo Scientific Q Exactive HF (LC-Hybrid Quadrupole-Orbitrap MS/MS) instrument using positive ion mode MS [50][51][52] . MS raw data were imported, processed, normalized, and reviewed using Progenesis QI v.2.1 (Non-linear Dynamics, Newcastle, UK). ...
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Assisted reproduction technologies for clinical and research purposes rely on a brief in vitro embryo culture which, despite decades of progress, remain suboptimal in comparison to the physiological environment. One promising tool to improve this technique is the development of bespoke microfluidic chambers. Here we present and validate a new microfluidic device in polydimethylsiloxane (PDMS) for the culture of early mouse embryos. Device material and design resulted embryo compatible and elicit minimal stress. Blastocyst formation, hatching, attachment and outgrowth formation on fibronectin-coated devices were similar to traditional microdrop methods. Total blastocyst cell number and allocation to the trophectoderm and inner cell mass lineages were unaffected. The devices were designed for culture of 10-12 embryos. Development rates, mitochondrial polarisation and metabolic turnover of key energy substrates glucose, pyruvate and lactate were consistent with groups of 10 embryos in microdrop controls. Increasing group size to 40 embryos per device was associated with increased variation in development rates and altered metabolism. Device culture did not perturb blastocyst gene expression but did elicit changes in embryo metabolome, which can be ascribed to substrate leaching from PDMS and warrant further investigation. This article is protected by copyright. All rights reserved.
... Each sample was then spun down to remove proteins and to aspirate the supernatant for analysis. Reverse-phase liquid chromatography was used, connected to a Thermo Scientific Q Exactive HF (LC-Hybrid Quadrupole-Orbitrap MS/MS) instrument using positive ion mode MS [26][27][28]. The data were thus imported, processed and normalized in Progenesis QI v.2.1 (Non-linear Dynamics, Newcastle, UK). ...
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Here we report the use of a microfluidic system to assess the differential metabolomics of murine embryos cultured with endometrial cells-conditioned media (CM). Groups of 10, 1-cell murine B6C3F1×B6D2F1 embryos were cultured in the microfluidic device. To produce CM, mouse uterine epithelial cells were cultured in potassium simplex optimized medium (KSOM) for 24 h. Media samples were collected from devices after 5 days of culture with KSOM (control) and CM, analyzed by reverse phase liquid chromatography and untargeted positive ion mode mass spectrometry analysis. Blastocyst rates were significantly higher (p < 0.05) in CM (71.8%) compared to control media (54.6%). We observed significant upregulation of 341 compounds and downregulation of 214 compounds in spent media from CM devices when compared to control. Out of these, 353 compounds were identified showing a significant increased abundance of metabolites involved in key metabolic pathways (e.g., arginine, proline and pyrimidine metabolism) in the CM group, suggesting a beneficial effect of CM on embryo development. The metabolomic study carried out in a microfluidic environment confirms our hypothesis on the potential of uterine epithelial cells to enhance blastocyst development. Further investigations are required to highlight specific pathways involved in embryo development and implantation.
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The members of the infant microbiome are governed by feeding choice (breastmilk vs. formula). Regardless of feeding choice, a competitive growth advantage can be provided to commensals through prebiotics ‐ either human milk oligosaccharides (HMOs) or plant oligosaccharides that are supplemented into formula. To characterize how prebiotics modulate commensal – pathogen interactions, we have designed and studied a minimal microbiome where a pathogen, Streptococcus agalactiae engages with a commensal, Streptococcus salivarius . We discovered that while S. agalactiae suppresses the growth of S. salivarius via increased lactic acid production, galacto‐oligosaccharides (GOS) supplementation reverses the effect. This result has major implications in characterizing how single species survive in the gut, what niche they occupy, and how they engage with other community members.
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Streptococcus agalactiae or Group B Streptococcus (GBS) is a gram‐positive bacterial pathobiont that is the etiological cause of severe perinatal infections. GBS can colonize the vagina of pregnant patients and invade tissues causing ascending infections of the gravid reproductive tract that lead to adverse outcomes including preterm birth, neonatal sepsis, and maternal or fetal demise. Additionally, transmission of GBS during labor or breastfeeding can also cause invasive infections of neonates and infants. However, human milk has also been shown to have protective effects against infection; a characteristic that is likely derived from antimicrobial and immunomodulatory properties of molecules that comprise human milk. Recent evidence suggests that human milk oligosaccharides (HMOs), short‐chain sugars that comprise 8‐20% of breast milk, have antimicrobial and anti‐biofilm activity against GBS and other bacterial pathogens. Additionally, HMOs have been shown to potentiate the activity of antibiotics against GBS. This review presents the most recent published work that studies the interaction between HMOs and GBS.
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Group B Streptococcus (GBS) is an important cause of disease in young infants, stillbirths, pregnant and post-partum women. GBS vaccines for maternal immunization are in development aiming to reduce this burden. Standardisation of case definitions and ascertainment methodologies for GBS disease is needed to support future trials of maternal GBS vaccines. Considerations presented here may also serve to promote consistency in observational studies and surveillance, to better establish disease burden. The World Health Organization convened a working group to provide consensus guidance for case ascertainment and case definitions of GBS disease in stillbirths, infants, pregnant and post-partum women, with feedback sought from external stakeholders. In intervention studies, case capture and case ascertainment for GBS disease should be based on antenatal recruitment of women, with active follow-up, systematic clinical assessment, standardised sampling strategies and optimised laboratory methods. Confirmed cases of invasive GBS disease in stillbirths or infants should be included in a primary composite endpoint for vaccine efficacy studies, with GBS cultured from a usually sterile body site (may be post-mortem). For additional endpoints, or observational studies, confirmed cases of GBS sepsis in pregnant and post-partum women should be assessed. Culture independent diagnostic tests (CIDTs) may detect additional presumed cases, however, the use of these diagnostics needs further evaluation. Efficacy of vaccination against maternal and neonatal GBS colonisation, and maternal GBS urinary tract infection could be included as additional, separate, endpoints and/or in observational studies. Whilst the focus here is on specific GBS disease outcomes, intervention studies also present an opportunity to establish the contribution of GBS across adverse perinatal outcomes, including all-cause stillbirth, preterm birth and neonatal encephalopathy.
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Background Group B Streptococcus (GBS) is the leading cause of neonatal sepsis and meningitis worldwide. We aimed to estimate the current burden of neonatal invasive GBS disease in the Netherlands, as a first step in providing an evidence base for policy makers on the potential benefits of a future maternal GBS vaccine. Methods Surveillance of neonatal invasive GBS occurs at the National Reference Laboratory for Bacterial Meningitis, where culture isolates from cerebrospinal fluid and blood are sent by diagnostic laboratories. From the number of cultures we estimated the incidence of neonatal (age 0–90 days) GBS meningitis and sepsis. We constructed a disease progression model informed by literature and expert consultation to estimate the disease burden of neonatal invasive GBS infection. As many neonates with a probable GBS sepsis are never confirmed by blood culture, we further estimated the disease burden of unconfirmed cases of probable GBS sepsis in sensitivity analyses. Results An estimated 97 cases and 6.5 deaths occurred in the Netherlands in 2017 due to culture positive neonatal invasive GBS infection. This incidence comprised 15 cases of meningitis and 42 cases of sepsis per 100.000 births, with an estimated mortality of 3.8 per 100.000 live births. A disease burden of 780 disability-adjusted life years (DALY) (95% CI 650–910) or 460 DALY per 100.000 live births was attributed to neonatal invasive GBS infection. In the sensitivity analysis including probable neonatal GBS sepsis the disease burden increased to 71 cases and 550 DALY (95% CI 460–650) per 100.000 live births. Conclusion In conclusion, neonatal invasive GBS infection currently causes a substantial disease burden in the Netherlands. However, important evidence gaps are yet to be filled. Furthermore, cases of GBS sepsis lacking a positive blood culture may contribute considerably to this burden potentially preventable by a future GBS vaccine.
Article
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Background: International guidelines lack a substantial consensus regarding management of asymptomatic full-term and late preterm neonates at risk for early-onset disease (EOS). Large cohorts of newborns are suitable to increase the understanding of the safety and efficacy of a given strategy. Methods: This is a prospective, area-based, cohort study involving regional birth facilities of Emilia-Romagna (Italy). We compared cases of EOS (at or above 35 weeks' gestation) registered in 2003-2009 (baseline period: 266,646 LBs) and in 2010-2016, after introduction of a new strategy (serial physical examinations, SPEs) for managing asymptomatic neonates at risk for EOS (intervention period: 265,508 LBs). Results: There were 108 cases of EOS (baseline period, n = 60; intervention period, n = 48). Twenty-two (20.4%) remained asymptomatic through the first 72 hours of life, whereas 86 (79.6%) developed symptoms, in most cases (52/86, 60.5%) at birth or within 6 hours. The median age at presentation was significantly earlier in the intrapartum antibiotic prophylaxis (IAP)-exposed than in the IAP-unexposed neonates (0 hours, IQR 0.0000-0.0000 vs 6 hours, IQR 0.0000-15.0000, p<0.001). High number of neonates (n = 531) asymptomatic at birth, exposed to intrapartum fever, should be treated empirically for each newborn who subsequently develops sepsis. IAP exposed neonates increased (12% vs 33%, p = 0.01), age at presentation decreased (median 6 vs 1 hours, p = 0.01), whereas meningitis, mechanical ventilation and mortality did not change in baseline vs intervention period. After implementing the SPEs, no cases had adverse outcomes due to the strategy, and no cases developed severe disease after 6 hours of life. Conclusions: Infants with EOS exposed to IAP developed symptoms at birth in almost all cases, and those who appeared well at birth had a very low chance of having EOS. The risk of EOS in neonates (asymptomatic at birth) exposed to intrapartum fever was low. Although definite conclusions on causation are lacking, our data support SPEs of asymptomatic newborns at risk for EOS. SPEs seems a safe and effective alternative to laboratory screening and empirical antibiotic therapy.
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We aimed to analyze the molecular characteristics, clonality and antimicrobial resistance profiles of group B streptococcus (GBS) isolates collected in Taiwan from invasive diseases and carriage. Multilocus sequence typing (MLST) was used to assess the genetic diversity of 225 GBS strains from neonates and adults with invasive GBS diseases. 100 GBS strains collected from colonized pregnant women during the same period were compared, and all strains were characterized for one of nine capsule genotypes. We also determined the susceptibilities of all GBS isolates to various antimicrobial agents. The most frequently identified serotypes that caused invasive disease in neonates were III (60.6%) and Ia (17.3%), whereas type VI (32.7%), Ib (19.4%), and V (19.4%) were the most common to cause invasive disease in adults. Serotype VI was the leading type that colonized pregnant women (35.0%). Twenty-six sequence types (STs) were identified, and 90.5% of GBS strains were represented by 6 STs. ST-17 and ST-1 were more prevalent in invasive diseases in neonates and adults, respectively. The majority of serotype III and VI isolates belonged to clonal complex (CC)-17 and CC-1, respectively. ST-17 strains were more likely to cause meningitis and late-onset disease than other strains. In addition, ST-12 and ST-17 GBS strains showed the highest rate of resistance to erythromycin and clindamycin (range: 75.8–100%). In conclusion, CC-17/type III and CC-1/type VI are the most important invasive pathogens in infants and non-pregnant adults in Taiwan, respectively. GBS genotypes vary between different age groups and geographical areas and should be considered during GBS vaccine development.
Article
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Background There is a lack of data regarding the prevalence of invasive group B streptococcus (GBS) infection among neonates in China. This study aimed to investigate the incidence and mortality of invasive GBS infection and to identify the risk factors in our hospital. Methods Seventy-four cases admitted between January 2011 and December 2016 was included in this study. A retrospective matched case-control study was conducted in a tertiary maternity and paediatric hospital. Risk factors for the acquisition of invasive GBS infection and mortality were analysed by univariable and multivariable analysis. Results We collected and analysed data from 74 infants aged < 3 months with invasive GBS infection. Among 67,985 live births, we calculated an incidence of 1.09 per 1000 live births (95%CI:0.81–1.37%); the incidence of Early-onset GBS disease (EOD, n = 65) and Late-onset GBS disease (LOD, n = 9) were 0.96‰(95%CI:0.73–1.19%) and 0.13‰(95%CI:0.04–0.22%), respectively. Overall, pneumonia accounted 63.1% (41/65) of EOD, and sepsis accounted 88.9% (8/9) cases of LOD, respectively. The overall case fatality rate was 8.11% (6/74), including 7.69% (5/65) among cases of EOD and 11.1% (1/9) among cases of LOD. No predictor of mortality was found. Membrane stripping (P = 0.005, aOR: 3.68, 95% CI: 1.48–9.13) and non-resident mother (P < 0.001, aOR: 5.88, 95% CI: 2.36–14.61) were independent risk factors for EOD; no increased risk was found for LOD. Conclusions This study demonstrates remarkable country-specific variation in comparison with other countries. Our findings can improve awareness of neonatal GBS infection and lay a cornerstone to ensure accurate representation of the burden.
Article
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Background: Maternal GBS colonization is associated with early-onset neonatal sepsis and extensive efforts are directed to preventing this complication. Less is known about maternal risks of GBS colonization. We seek to provide a modern estimate of the incidence and impact of maternal GBS colonization and invasive GBS disease. Methods: A single center historical cohort study of all births between 2003 and 2015 was performed. Data was collected via electronic health record abstraction using an institutional specific tool. Descriptive statistics were performed regarding GBS status. Inferential statistics were performed comparing risk of adverse pregnancy outcomes in cohorts with and without GBS colonization as well as cohorts with GBS colonization and invasive GBS disease. Results: A total of 60,029 deliveries were included for analysis. Overall, 21.6% of the population was GBS colonized and 0.1% had invasive GBS disease. GBS colonization was associated with younger maternal age, Black race, non-Hispanic ethnicity, chronic hypertension, preexisting diabetes, and tobacco use (p<0.01). In the adjusted analyses, there was an increased risk of gestational diabetes (aRR 1.21, 95% CI 1.11-1.32) in colonized pregnancies and a decreased incidence of short cervix (aRR 0.64, 95% CI 0.52-0.79), chorioamnionitis (aRR 0.76, 95% CI 0.66-0.87), wound infection (aRR 0.75, 95% CI 0.64-0.88), and operative delivery (aRR 0.85, 95% CI 0.83-0.88). Conclusions: This modern-day large cohort of all births over a 12-year period demonstrates a GBS colonization rate of 21.6%. This data reflects a need to assess maternal and perinatal outcomes in addition to neonatal GBS sepsis rates to inform decisions regarding the utility of maternal vaccination.
Article
2′-Fucosyllactose (2′-FL) is a ubiquitous oligosaccharide in human milk. Importantly, this carbohydrate promotes the growth of several strains of Bifidobacteria, a class of beneficial gut commensal, and inhibits epithelial binding of pathogens. In light of these protective effects, we elected to evaluate the potential of 2′-FL to serve as an antibacterial agent against Group B Streptococcus (GBS). While 2′-FL was devoid of any substantial antimicrobial or antibiofilm activity, conversion of 2′-FL to its reducing end β-amine provided a novel antibiofilm compound.
Article
ConspectusThis Account describes the risky proposition of organizing a multidisciplinary team to interrogate a challenging problem in chemical biology: characterizing how human milk, at the molecular level, protects infants from infectious diseases. At the outset, our initial hypothesis was that human milk oligosaccharides (HMOs) possess antimicrobial and antivirulence activities. Early on, we discovered that HMOs do indeed modulate bacterial growth and biofilm production for numerous bacterial pathogens. In light of this discovery, three priorities emerged for our program moving forward. The first was to decode the mode of action behind this activity. The second was to decipher the functional effects of HMO structural diversity as there are ca. 200 unique HMOs present in human milk. Finally, we set our sights on discovering novel uses for HMOs as we believed this would uniquely position our team to achieve a major breakthrough in human health and wellness.Through a combination of fractionation techniques, chemical synthesis, and industrial partnerships, we have determined the identities of several HMOs with potent antimicrobial activity against the important neonate pathogen Group B Streptococcus (Group B Strep; GBS). In addition to a structure-activity relationship (SAR) study, we observed that HMOs are effective adjuvants for intracellular-targeting antibiotics against GBS. This included two antibiotics that GBS has evolved resistance to. At their half maximal inhibitory concentration (IC 50 ), heterogeneous HMOs reduced the minimum inhibitory concentration (MIC) of select antibiotics by up to 32-fold. Similarly, we observed that HMOs potentiate the activity of polymyxin B (Gram-negative-selective antibiotic) against GBS (Gram-positive species). Based on these collective discoveries, we hypothesized that HMOs function by increasing bacterial cell permeability, which would be a novel mode of action for these molecules. This hypothesis was validated as HMOs were found to increase membrane permeability by around 30% compared to an untreated control. The question that remains is how exactly HMOs interact with bacterial membranes to induce permeability changes (i.e., through promiscuous insertion into the bilayer, engagement of proteins involved in membrane synthesis, or HMO-capsular polysaccharide interactions). Our immediate efforts in this regard are to apply chemoproteomics to identify the molecular target(s) of HMOs. These investigations are enabled through manipulation of HMOs produced via total synthesis or enzymatic and whole-cell microbial biotransformation.
Article
Cigarette smoking is associated with chronic obstructive pulmonary disease and chronic bronchitis. Acquired ion transport abnormalities, including cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction, caused by cigarette smoking have been proposed as potential mechanisms for mucus obstruction in chronic bronchitis. Although e-cigarette use is popular and perceived to be safe, whether it harms the airways via mechanisms altering ion transport remains unclear. In the present study, we sought to determine if e-cigarette vapor, like cigarette smoke, has the potential to induce acquired CFTR dysfunction, and to what degree. Electrophysiological methods demonstrated reduced chloride transport caused by vaporized e-cigarette liquid or vegetable glycerin at various exposures (30 min, 57.2% and 14.4% respectively, vs. control; P < 0.0001), but not by unvaporized liquid (60 min, 17.6% vs. untreated), indicating that thermal degradation of these products is required to induce the observed defects. We also observed reduced ATP-dependent responses (-10.8 ± 3.0 vs. -18.8 ± 5.1 μA/cm2 control) and epithelial sodium channel activity (95.8% reduction) in primary human bronchial epithelial cells after 5 minutes, suggesting that exposures dramatically inhibit epithelial ion transport beyond CFTR, even without diminished transepithelial resistance or cytotoxicity. Vaporizing e-cigarette liquid produced reactive aldehydes, including acrolein (shown to induce acquired CFTR dysfunction), as quantified by mass spectrometry, demonstrating that respiratory toxicants in cigarette smoke can also be found in e-cigarette vapor (30 min air, 224.5 ± 15.99; unvaporized liquid, 284.8 ± 35.03; vapor, 54,468 ± 3,908 ng/ml; P < 0.0001). E-cigarettes can induce ion channel dysfunction in airway epithelial cells, partly through acrolein production. These findings indicate a heretofore unknown toxicity of e-cigarette use known to be associated with chronic bronchitis onset and progression, as well as with chronic obstructive pulmonary disease severity.
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For newborns, human milk oligosaccharides (HMOs) serve as an important source of protection against pathogenic Gram-positive and Gram-negative bacteria. HMOs most notably prevent infection by functionung as decoy receptors that bind pathogens to prevent cellular adhesion. HMOs also play a protective role by acting as prebiotics that selectively promote the growth of symbiotic gut bacteria over pathogenic species. Fucosylated HMOs in particular are well-known for their roles as both decoy receptors and prebiotics. Recently, we discovered that HMOs possess antimicrobial activity against Group B Streptococcus (GBS) by increasing cellular permeability. As HMO extracts from a single donor can contain over 100 different structures, however, studies using heterogenous HMO mixtures do not provide insight into the specific structural requirements needed to achieve antimicrobial activity. In this study, we address this void by completing a structure activity study on the antimicrobial and antibiofilm activities of 6 neutral, fucosylated and 5 neutral, nonfucosylated HMOs against GBS. We determined that while the presence of fucose alone does not correlate to antimicrobial activity, the location and degree of fucosylation does play a key role in HMO antimicrobial activity. Moreover, the antimicrobial and antibiofilm activities of single HMOs were found to be strain-specific. This further supports our vision of developing narrow-spectrum antibacterial agents against GBS.