Carriage and within-host diversity of mcr-1.1-harboring Escherichia coli from pregnant mothers: inter- and intra-mother transmission dynamics of mcr-1.1

Exchange of antimicrobial resistance genes via mobile genetic elements occur in the gut which can be transferred from mother to neonate during birth. This study is the first to analyze transmissible colistin resistance gene, mcr, in pregnant mothers and neonates. Samples were collected from pregnant mothers (rectal) and septicaemic neonates (rectal & blood) and analyzed for presence of mcr, its transmissibility, genome diversity, and exchange of mcr between isolates within an individualand across different individuals (not necessarily mother-baby pairs). mcr-1.1 was detected in rectal samples of pregnant mothers (n=10, 0.9%), but not in neonates. All mcr-positive mothers gave birth to healthy neonates from whom rectal specimen were not collected. Hence, transmission of mcr between these mother-neonate pairs could not be studied. mcr-1.1 was noted only in Escherichia coli (phylogroup A & B1), and carried few resistance and virulence genes. Isolates belonged to diverse sequence types (n=11) with two novel STs (ST12452, ST12455). mcr-1.1 was borne on conjugative IncHI2 bracketed between ISApl1 on Tn6630, and the plasmids exhibited similarities in sequences across the study isolates. Phylogenetic comparison showed that study isolates were related to mcr-positive isolates of animal origin from Southeast Asian countries. Spread of mcr-1.1 within this study occurred either via similar mcr-positive clones or similar mcr-bearing plasmids in mothers. Though this study could not build evidence for mother-baby transmission, but presence of such genes in the maternal specimen may enhance the chances of transmission to neonates

Dopamine Regulates Angiogenesis in Normal Dermal Wound Tissues

Cutaneous wound healing is a normal physiological process and comprises different phases. Among these phases, angiogenesis or new blood vessel formation in wound tissue plays an important role. Skin is richly supplied by sympathetic nerves and evidences indicate the significant role of the sympathetic nervous system in cutaneous wound healing. Dopamine (DA) is an important catecholamine neurotransmitter released by the sympathetic nerve endings and recent studies have demonstrated the potent anti-angiogenic action of DA, which is mediated through its D2 DA receptors. We therefore postulate that this endogenous catecholamine neurotransmitter may have a role in the neovascularization of dermal wound tissues and subsequently in the process of wound healing. In the present study, the therapeutic efficacy of D2 DA receptor antagonist has been investigated for faster wound healing in a murine model of full thickness dermal wound. Our results indicate that treatment with specific D2 DA receptor antagonist significantly expedites the process of full thickness normal dermal wound healing in mice by inducing angiogenesis in wound tissues. The underlined mechanisms have been attributed to the up-regulation of homeobox transcription factor HoxD3 and its target α5β1 integrin, which play a pivotal role in wound angiogenesis. Since D2 DA receptor antagonists are already in clinical use for other disorders, these results have significant translational value from the bench to the bedside for efficient wound management along with other conventional treatment modalities

Dopamine Regulates Mobilization of Mesenchymal Stem Cells during Wound Angiogenesis

Angiogenesis is an important step in the complex biological and molecular events leading to successful healing of dermal wounds. Among the different cellular effectors of wound angiogenesis, the role of mesenchymal stem cells (MSCs) is of current interest due to their transdifferentiation and proangiogenic potentials. Skin is richly innervated by sympathetic nerves which secrete dopamine (DA) and we have recently shown that concentration of DA present in synaptic cleft can significantly inhibit wound tissue neovascularization. As recent reports indicate that MSCs by mobilizing into wound bed play an important role in promoting wound angiogenesis, we therefore investigated the effect of DA on the migration of MSCs in wound tissues. DA acted through its D2 receptors present in the MSCs to inhibit their mobilization to the wound beds by suppressing Akt phosphorylation and actin polymerization. In contrast, this inhibitory effect of DA was reversed after treatment with specific DA D2 receptor antagonist. Increased mobilization of MSCs was demonstrated in the wound site following blockade of DA D2 receptor mediated actions, and this in turn was associated with significantly more angiogenesis in wound tissues. This study is of translational value and indicates use of DA D2 receptor antagonists to stimulate mobilization of these stem cells for faster regeneration of damaged tissues

Neonatal sepsis and mortality in low-income and middle-income countries from a facility-based birth cohort: an international multisite prospective observational study

Background Neonatal sepsis is a primary cause of neonatal mortality and is an urgent global health concern, especially within low-income and middle-income countries (LMICs), where 99% of global neonatal mortality occurs. The aims of this study were to determine the incidence and associations with neonatal sepsis and all-cause mortality in facility-born neonates in LMICs. Methods The Burden of Antibiotic Resistance in Neonates from Developing Societies (BARNARDS) study recruited mothers and their neonates into a prospective observational cohort study across 12 clinical sites from Bangladesh, Ethiopia, India, Pakistan, Nigeria, Rwanda, and South Africa. Data for sepsis-associated factors in the four domains of health care, maternal, birth and neonatal, and living environment were collected for all mothers and neonates enrolled. Primary outcomes were clinically suspected sepsis, laboratory-confirmed sepsis, and all-cause mortality in neonates during the first 60 days of life. Incidence proportion of livebirths for clinically suspected sepsis and laboratory-confirmed sepsis and incidence rate per 1000 neonate-days for all-cause mortality were calculated. Modified Poisson regression was used to investigate factors associated with neonatal sepsis and parametric survival models for factors associated with all-cause mortality. Findings Between Nov 12, 2015 and Feb 1, 2018, 29 483 mothers and 30 557 neonates were enrolled. The incidence of clinically suspected sepsis was 166·0 (95% CI 97·69–234·24) per 1000 livebirths, laboratory-confirmed sepsis was 46·9 (19·04–74·79) per 1000 livebirths, and all-cause mortality was 0·83 (0·37–2·00) per 1000 neonate-days. Maternal hypertension, previous maternal hospitalisation within 12 months, average or higher monthly household income, ward size (>11 beds), ward type (neonatal), living in a rural environment, preterm birth, perinatal asphyxia, and multiple births were associated with an increased risk of clinically suspected sepsis, laboratory-confirmed sepsis, and all-cause mortality. The majority (881 [72·5%] of 1215) of laboratory-confirmed sepsis cases occurred within the first 3 days of life. Interpretation Findings from this study highlight the substantial proportion of neonates who develop neonatal sepsis, and the high mortality rates among neonates with sepsis in LMICs. More efficient and effective identification of neonatal sepsis is needed to target interventions to reduce its incidence and subsequent mortality in LMICs. Funding Bill & Melinda Gates Foundation

Effects of antibiotic resistance, drug target attainment, bacterial pathogenicity and virulence, and antibiotic access and affordability on outcomes in neonatal sepsis: an international microbiology and drug evaluation prospective substudy (BARNARDS)

Background Sepsis is a major contributor to neonatal mortality, particularly in low-income and middle-income countries (LMICs). WHO advocates ampicillin–gentamicin as first-line therapy for the management of neonatal sepsis. In the BARNARDS observational cohort study of neonatal sepsis and antimicrobial resistance in LMICs, common sepsis pathogens were characterised via whole genome sequencing (WGS) and antimicrobial resistance profiles. In this substudy of BARNARDS, we aimed to assess the use and efficacy of empirical antibiotic therapies commonly used in LMICs for neonatal sepsis. Methods In BARNARDS, consenting mother–neonates aged 0–60 days dyads were enrolled on delivery or neonatal presentation with suspected sepsis at 12 BARNARDS clinical sites in Bangladesh, Ethiopia, India, Pakistan, Nigeria, Rwanda, and South Africa. Stillborn babies were excluded from the study. Blood samples were collected from neonates presenting with clinical signs of sepsis, and WGS and minimum inhibitory concentrations for antibiotic treatment were determined for bacterial isolates from culture-confirmed sepsis. Neonatal outcome data were collected following enrolment until 60 days of life. Antibiotic usage and neonatal outcome data were assessed. Survival analyses were adjusted to take into account potential clinical confounding variables related to the birth and pathogen. Additionally, resistance profiles, pharmacokinetic–pharmacodynamic probability of target attainment, and frequency of resistance (ie, resistance defined by in-vitro growth of isolates when challenged by antibiotics) were assessed. Questionnaires on health structures and antibiotic costs evaluated accessibility and affordability. Findings Between Nov 12, 2015, and Feb 1, 2018, 36 285 neonates were enrolled into the main BARNARDS study, of whom 9874 had clinically diagnosed sepsis and 5749 had available antibiotic data. The four most commonly prescribed antibiotic combinations given to 4451 neonates (77·42%) of 5749 were ampicillin–gentamicin, ceftazidime–amikacin, piperacillin–tazobactam–amikacin, and amoxicillin clavulanate–amikacin. This dataset assessed 476 prescriptions for 442 neonates treated with one of these antibiotic combinations with WGS data (all BARNARDS countries were represented in this subset except India). Multiple pathogens were isolated, totalling 457 isolates. Reported mortality was lower for neonates treated with ceftazidime–amikacin than for neonates treated with ampicillin–gentamicin (hazard ratio [adjusted for clinical variables considered potential confounders to outcomes] 0·32, 95% CI 0·14–0·72; p=0·0060). Of 390 Gram-negative isolates, 379 (97·2%) were resistant to ampicillin and 274 (70·3%) were resistant to gentamicin. Susceptibility of Gram-negative isolates to at least one antibiotic in a treatment combination was noted in 111 (28·5%) to ampicillin–gentamicin; 286 (73·3%) to amoxicillin clavulanate–amikacin; 301 (77·2%) to ceftazidime–amikacin; and 312 (80·0%) to piperacillin–tazobactam–amikacin. A probability of target attainment of 80% or more was noted in 26 neonates (33·7% [SD 0·59]) of 78 with ampicillin–gentamicin; 15 (68·0% [3·84]) of 27 with amoxicillin clavulanate–amikacin; 93 (92·7% [0·24]) of 109 with ceftazidime–amikacin; and 70 (85·3% [0·47]) of 76 with piperacillin–tazobactam–amikacin. However, antibiotic and country effects could not be distinguished. Frequency of resistance was recorded most frequently with fosfomycin (in 78 isolates [68·4%] of 114), followed by colistin (55 isolates [57·3%] of 96), and gentamicin (62 isolates [53·0%] of 117). Sites in six of the seven countries (excluding South Africa) stated that the cost of antibiotics would influence treatment of neonatal sepsis

Dopamine (DA) through its D₂ receptors inhibits VEGF induced migration of BM-MSCs by regulating phosphorylation of VEGFR-2 and Akt and actin polymerization.

<p>(<b>A and B</b>) Effect of DA on VEGF induced phosphorylation of VEGFR-2 and Akt in murine MSCs. Lane 1: Cells stimulated with VEGF (10 ng/ml). Lane 2: Cells pretreated with 1 µM DA before being exposed to VEGF (10 ng/ml). Lane 3: Cells treated with 100 µM eticlopride followed by DA and VEGF. Addition of DA significantly inhibited VEGF induced phosphorylation of both VEGFR-2 receptors and its downstream target Akt when compared with VEGF treated control. Pre-treatment with eticlopride, abrogated DA induced dephosphorylation of both VEGFR-2 and Akt. However, expression of total VEGFR-2 and total Akt remained unchanged. Results are representative of six separate experiments each yielding similar results. (<b>C</b>) In actin polymerization assay, VEGF induced MSCs showed significant polymerization of actin cytoskeleton leading to formation of F-actin. However, no such alterations were observed in case of Akt silenced MSCs which showed strikingly lower migratory activity against VEGF than normal MSCs. (<b>D</b>) Effect of DA on VEGF induced actin polymerization in MSCs. Treatment with 1 µM DA had significant inhibitory effect on actin polymerization dynamics in MSCs when compared to VEGF treated control. However, pre-treatment with specific DA D₂ receptor antagonist eticlopride (100 µM) abrogated the DA induced changes in the actin polymerization dynamics of the VEGF induced MSCs. Original magnifications, ×1000.</p

Dopamine (DA) D₂ receptor antagonist treatment stimulate incorporation of MSCs into neovessels of wound bed and subsequent angiogenesis in wound bed.

<p>(<b>A</b>) Effect of eticlopride treatment on incorporation of transplanted MSCs into newly formed blood vessels in wound bed. MSCs were labeled with CM-Dil and injected into the tail vein of both control and eticlopride treated back skin-injured mice. At day 6, the skin from the wound area was collected and sectioned with cryomicrotome. Frozen sections were immunostained with anti-CD31 antibody and FITC-conjugated secondary antibody and analyzed to determine the extent of incorporation of transplanted MSCs into blood vessels. Here co-localization study showed that exogenously transplanted MSCs, labeled with fluorescent dye CM-Dil (red in colour), had integrated into the newly formed blood vessels (green in colour) in wound site in much greater number following DA D₂ receptor antagonist treatment than vehicle treated controls. Original magnifications, ×200. (<b>B</b>) Effect of MSC transplantation on formation of new blood vessels in wound bed. Immunohistochemical staining of CD31, a specific endothelial cell surface marker, shows significantly greater number of microvessels in wound tissue sections of DA D₂ receptor antagonist treated mice in comparison to vehicle treated controls at day 6 post wounding. Treatment with MSCs significantly increases microvessel density in wound bed in comparison to saline treated controls. However, eticlopride treatment along with MSCs transplantation is most effective in increasing angiogenesis in wound tissue. Original magnifications ×200. (<b>C</b>) Graphical representation showing microvessels density in wound tissue sections of different experimental groups at day 6 after post wounding (*, P<0.05). Microvessel density (CD31 positive cells) in wound bed was measured by counting the number of microvessels in 10 randomly chosen high power microscopic fields within the sections.</p

Treatment with dopamine (DA) D₂ receptor antagonist significantly accelerated mobilization of exogenously transplanted MSCs towards wound bed.

<p>(<b>A</b>) Flow cytometric analysis of DA D₂ receptors in murine MSCs. To confirm that CD34⁻ CD45⁻ CD105⁺ SSEA-4⁺ cells express DA D₂ receptors, <i>in vitro</i> expanded Lin^neg bone marrow cell population (containing CD45⁻, CD11b⁻ cell populations) were initially gated to exclude dead cells and debris and then the CD34⁻ cell population were selected and these CD34⁻ CD45⁻ cells were evaluated for presence of CD105, SSEA-4 and DA D₂ receptors. Results showed that almost 86% cells of the total MSC population (both CD34⁻ CD45⁻ CD105⁺ cells and CD34⁻ CD45⁻ SSEA-4⁺ cells) express DA D₂ receptors on their surfaces. (<b>B</b>) Western blot analysis of DA D₂ receptors in murine BM-MSCs. H4 neuroglioma cell line and sarcoma-180 (S-180) tumor cells were used as positive and negative controls, respectively. (<b>C</b>) Effect of DA D₂ receptor antagonist treatment on mobilization of exogenously transplanted MSCs towards wound site. MSCs were labeled with BrdU in culture and injected into the tail vein of both control and eticlopride treated back skin-injured mice. After completion of eticlopride treatment, at 6th day the skin from the wound area was collected and analyzed by immunohistochemistry using BrdU labeling and detection kit (Roche Applied Science). Significantly higher number of BrdU positive transplanted MSCs are located in the wound bed of eticlopride treated group than vehicle treated control group, showing DA D₂ receptor antagonist treatment has significant positive effect on mobilization of exogenously transplanted MSCs towards wound site. Original magnifications, ×200. Results are representative of six separate experiments each yielding similar results. (<b>D</b>) Graphical representation showing significantly higher number of exogenously transplanted MSCs (BrdU positive cells) in wound bed of eticlopride treated groups compared to vehicle treated controls at day 6 post wounding (*, P<0.05). Number of transplanted MSCs cells was measured by counting the number of BrdU positive cells in 10 randomly chosen high power microscopic fields within the sections.</p

VEGF regulates migration of murine BM-MSCs through activation of Akt.

<p>(<b>A</b>) Effects of VEGF on <i>in vitro</i> migration of MSCs in transwell chambers. After incubation with MSCs at 37°C for 24 hours, VEGF was added. After overnight incubation cells remaining on the upper face of the filters were removed with a cotton wool swab and migrated cells that remained on the lower face of the filters were stained with Gill's Haematoxylin, counted and photographed. Numbers of migrated cells were counted in 10 high-power fields (HPFs) after subtraction of the basal migration observed in the presence of DMEM alone (without presence of any growth factor). Dopamine (DA) significantly inhibits VEGF induced migration of MSCs (*, P<0.05), whereas when cells were pre-treated with 100 µM eticlopride (a specific DA D₂ receptor antagonist), the inhibitory effect of 1 µM DA on VEGF mediated mobilization was abrogated. Results are representative of six separate experiments each yielding similar results. (<b>B</b>) Silencing of Akt in MSCs by siRNA transfection. After 48 hours of transfection, no expression of Akt was found in MSCs transfected with Akt siRNA (iAkt) whereas control MSCs in which control siRNA containing scrambled sequence (iScr) was transfected showed similar expression of Akt as normal non-transfected MSCs. (<b>C</b>) Effect of Akt silencing on VEGF induced migration of MSCs in a chemotaxis assay. Numbers of migrated cells were counted in 10 high-power fields (HPFs) after subtraction of the basal migration observed in the presence of DMEM alone (without presence of any growth factor). Silencing of Akt significantly inhibits VEGF induced migration of MSCs (*, P<0.05). Results are representative of six separate experiments each yielding similar results. (<b>D</b>) Flow cytometric analysis of VEGFR-2 receptors in murine MSCs. To confirm that CD34⁻ CD45⁻ CD105⁺ cells express VEGFR-2 receptors, CD34⁻ cells were selected from <i>in vitro</i> expanded Lin^neg bone marrow cell population (containing CD45⁻, CD11b⁻ cell populations) and these CD34⁻ CD45⁻ cells were evaluated for presence of CD105 and VEGFR-2 receptors. Results showed that almost 89% cells of the total MSC population (CD34⁻ CD45⁻ CD105⁺ cells) express VEGFR-2 receptors on their surfaces.</p