A lone member of one of these groups, and a phylogenetic outlier,

A lone member of one of these groups, and a phylogenetic outlier, is the T6SS of F. tularensis, a highly virulent Gram-negative intracellular pathogen, which causes the zoonotic disease tularemia in humans and many mammals [8]. The T6SS is encoded by a 33-kb gene cluster, the Francisella pathogenicity island (FPI), which comprises 17-20 genes that form

a secretion system that secretes up to 8 FPI-encoded substrates during intramacrophage infection [9–11]. Studies on FPI mutants have revealed that bacteria replicate only after phagosomal escape and, thus, mutants that are incapable of escape show a null phenotype with lack of intracellular growth, no cytopathogenic effects, LY2874455 molecular weight and avirulence in experimental models [12–19]. In addition, uptake of F. tularensis bacteria leads to rapid induction of a proinflammatory response, which is repressed

upon bacterial internalization via modulation of host cell signaling and, again, execution of mTOR inhibitor these mechanisms appears to require a cytosolic localization of bacteria [17, 19–22]. A majority of FPI mutants have shown dichotomous phenotypes also in this respect and the mutants that are unable to escape from the phagosome do not repress of host cell signaling, whereas other mutants show the same phenotypes as the parental strains [19, 22]. Two notable exceptions are the ΔiglI and ΔiglG mutants of LVS, since these are avirulent but show intact growth in certain monocytic cells, although with only marginal cytopathogenic effects [17]. An FPI protein of special interest is PdpC, since a truncated form of the protein has been identified in FSC043, an attenuated, spontaneous mutant of the prototypic F. tularensis subspecies tularensis strain SCHU S4 [23]. We have previously characterized the FSC043 strain and observed that it displays impaired replication Astemizole in murine monocytic cells [24]. Therefore, we hypothesized that the spontaneous

mutation could be related to the impaired intracellular replication of the mutant. In the present study, we generated and characterized a ΔpdpC mutant of F. tularensis LVS. We observed a phenotype that was distinct from all previously described FPI mutants, since it showed very impaired phagosomal escape and lack of intramacrophage replication, but still pronounced cytopathogenic effects, although distinct from those of the parental strain. Results In silico analyses and localization of PdpC To characterize PdpC, in silico analyses together with cell fractionation were carried out. PdpC was predicted to be a cytoplasmic 156-kDa protein with putative transmembrane regions.

Similar results were not found on the skin for any time points (F

Similar results were not found on the skin for any time points (Figure 3, Panels B and C, and Additional file 2: Figures S4 and S5). We did not control for skin-related hygiene practices, which may have affected the skin microbiota.

Figure 3 Conservation of CRISPR spacer content by time of day sampled. Each panel demonstrates the relative conservation of spacers (±standard deviation) within the morning time points for each subject (M vs. M), comparisons of the morning time points with this website noon time points (M vs. N), and comparisons of the morning time points with the evening time points (M vs E) for subject #1 (magenta), subject #2 [22], subject #3 (red), and subject #4 (cyan). Panels A and B represent salivary SGII and SGI CRISPR spacers, respectively. Panels C and D represent skin-derived

SGII and SGI CRISPR spacers, respectively. The ‘*’ represents subjects in which the relative conservation of spacers for the morning time points is significantly (p ≤ 0.05) greater than for comparisons of morning and noon/evening time points. When compared to skin spacers, the proportion of shared spacers in saliva over time in each subject was highly significant (p < 0.005 in all subjects for SGII and SGI spacers) (Additional file 1: Table S4). In some cases there were more shared spacers between skin and saliva than there were for comparisons of different find more time points within the skin of the same subject for SGII spacers (44% shared between saliva and skin versus 37% shared in skin for Subject #1; 41% vs 36% in Subject #2; 11% vs 15% for Subject #3; 25% vs 24% for Subject #4) and for SGI spacers (42% shared between saliva and skin versus 39% shared in skin for Subject #1; 30% vs 28% in Subject #2; 16% vs 10% for Subject #3; 37% vs 36% for Subject #4). These data demonstrate Bcl-w a smaller group of shared spacers present on the skin of these subjects than in their saliva, which suggests greater heterogeneity in the skin microbiota. We also examined

spacers shared between different subjects and whether there were any SGI CRISPR spacers shared with SGII spacers. On average, 21.86 ± 1.98% of the SGI spacers were shared between subjects, 20.93 ± 2.34% of the SGII spacers were shared between subjects, while only 0.011 ± 0.004% (p < 0.001) of the SGI and SGII spacers were shared between subjects, indicating that either SGI and SGII spacers likely target different viruses/plasmids, or target different portions of the same viruses/plasmids [37]. CRISPR locus assembly Because of the short read lengths of most of the sequences produced in this study, CRISPR loci could not be assembled; however, longer reads sequenced from the day 14 AM sample from subject #3 could be assembled into loci.

Figure 2 PCR-DGGE analysis with Lactobacillus-specific primers A

Figure 2 PCR-DGGE analysis with Lactobacillus-specific primers. Analysis Selleck Adriamycin was conducted on the vaginal samples collected at 33rd (W33) and 37th (W37) week of gestation from 15 women supplemented with the probiotic VSL#3 [(P) N. 1–15] and 12 control women [(C) N. 16–27]. N: woman number; W: week of

gestation; T: type of supplementation. (A) PCR-DGGE fingerprints. M, external reference marker. Band L16 corresponds to L. helveticus (GenBank accession number: AB571603) (B) Dendrogram of the DGGE profiles shown in panel A. Pearson correlation was used to calculate the similarity in DGGE profiles. Richness indexes ranged from 5.7 (W33) to 5.4 (W37) for P group and from 6.3 (W33) to 6.8 (W37) for C group. Mean values of SI were 79% and 80% for

P and C groups, respectively (Table 1). Only 2 women included in P group showed SIs < 50% (N. 1 and 15). Wilcoxon Signed Rank selleck compound Test highlighted significant differences between DGGE profiles related to W33 and W37 for women N. 7 and 10, accounting for 13% of women included in P group. Comparing this percentage with the 33% obtained by DGGE analysis with HDA1-GC/HDA2 primer set, the probiotic intake seemed to have a more extended impact on total bacteria than lactobacilli. Notably, only for woman N. 10, significant differences were found between W33- and W37-related DGGE patterns Tolmetin for both HDA1-GC/HDA2 and Lac1/Lac2-GC primer sets. The peak height analysis by Wilcoxon Signed Rank Test allowed us to identify a band, denominated L16 (Figure 2), which significantly changed after probiotic supplementation. Sequencing of the DNA extracted from this band revealed 100% homology with L. helveticus strains. The nucleotide sequence of this DGGE fragment was deposited in DDBJ Nucleotide Sequence Database under the accession number AB571603. L. helveticus was found to be a representative species within lactobacilli

population since it was detected in 9 women supplemented with VSL#3 and 2 control women, corresponding to a frequency of occurrence of 40.7%. Notably, a general decrease in the intensity of L. helveticus band was observed in P group while no variations were appreciable in C group. Cluster analysis showed that Lactobacillus-specific DGGE profiles related to the time points W33 and W37 were closely related for all control women and for the majority of women administered with VSL#3, except for the subjects N. 1 and 15 (Figure 2). Quantitative variations of vaginal bacterial populations Quantitative real-time PCR (qPCR) was performed to analyze changes in concentration of Lactobacillus, Bifidobacterium and Streptococcus thermophilus, that were included in the probiotic VSL#3, and Gardnerella vaginalis, Atopobium, Prevotella and Veillonella, that are important BV-related genera and species [22, 28].

J Bacteriol 2007,189(23):8405–8416 CrossRefPubMed 18 Shelburne S

J Bacteriol 2007,189(23):8405–8416.CrossRefPubMed 18. Shelburne SA III, Keith D, Horstmann N, Sumby P, Davenport MT, Graviss EA, Brennan RG, Musser JM: A direct link between carbohydrate utilization and virulence in the major human pathogen group A Streptococcus. Proc Natl Acad Sci USA 2008,105(5):1698–1703.CrossRefPubMed 19. Wen ZT, Burne RA: Functional genomics approach to identifying genes required for biofilm development by Streptococcus mutans. Appl Environ Microbiol 2002,68(3):1196–1203.CrossRefPubMed 20. Bizzini A, Entenza JM, Moreillon

P: Loss of penicillin tolerance by inactivating the carbon catabolite repression determinant CcpA in Streptococcus gordonii. J Antimicrob Chemother 2007,59(4):607–615.CrossRefPubMed 21. De Lencastre H, Wu SW, Pinho MG, Ludovice AM, Filipe S, Gardete S, Sobral R, Gill S, Chung M, Tomasz A: Antibiotic resistance as a stress response: complete sequencing Sirtuin activator inhibitor of a large number of chromosomal loci in Staphylococcus aureus strain COL that impact on the expression of resistance to methicillin. Microb Rabusertib solubility dmso Drug Resist 1999,5(3):163–175.CrossRefPubMed 22. Seidl K, Bischoff M, Berger-Bächi B: CcpA mediates the catabolite repression of tst in Staphylococcus aureus. Infect Immun 2008,76(11):5093–5099.CrossRefPubMed 23. Seidl K, Goerke C, Wolz C, Mack D, Berger-Bächi B, Bischoff M: The Staphylococcus aureus CcpA affects biofilm formation. Infect

Immun 2008,76(5):2044–2050.CrossRefPubMed EGFR inhibiton 24. Seidl K, Stucki M, Rüegg M, Goerke C, Wolz C, Harris L, Berger-Bächi B,

Bischoff M:Staphylococcus aureus CcpA affects virulence determinant production and antibiotic resistance. Antimicrob Agents Chemother 2006,50(4):1183–1194.CrossRefPubMed 25. Sezonov G, Joseleau-Petit D, D’Ari R:Escherichia coli physiology in Luria-Bertani broth. J Bacteriol 2007, 189:8746–8749.CrossRefPubMed 26. Database of the Genomes Annotated at Nite (DOGAN)[http://​www.​bio.​nite.​go.​jp/​dogan/​MicroTop?​GENOME_​ID=​n315] 27. Kanehisa M: A database for post-genome analysis. Trends Genet 1997,13(9):375–376.CrossRefPubMed 28. Oskouian B, Stewart GC: Repression and catabolite repression of the lactose operon of Staphylococcus aureus. J Bacteriol 1990,172(7):3804–3812.PubMed 29. Oskouian B, Stewart G: Cloning and characterization of the repressor gene of the Staphylococcus aureus lactose operon. J Bacteriol 1987,169(12):5459–5465.PubMed 30. Blumenthal HJ: Glucose catabolism in Staphylococci. Staphylococci (Edited by: Cohen JO). New York: Wiley-Intersience 1972, 111–135. 31. Scovill W, Schreier H, Bayles K: Identification and characterization of the pckA gene from Staphylococcus aureus. J Bacteriol 1996,178(11):3362–3364.PubMed 32. Blencke H-M, Homuth G, Ludwig H, Mader U, Hecker M, Stülke J: Transcriptional profiling of gene expression in response to glucose in Bacillus subtilis : regulation of the central metabolic pathways. Metab Eng 2003,5(2):133–149.CrossRefPubMed 33.

The persistence seen with the subject-specific LAB strains cultiv

The persistence seen with the subject-specific LAB strains cultivated from faeces is also interesting in this regard. Commercialisation of LAB strains for probiotic use is dependent on a number of factors, however, from our study and other work, it appears that many commercialised LAB strains are genotypically identical to reference strains deposited in recognised culture collections (Table 2). The

fingerprinting strategy described herein could be used to select LAB GDC-0973 nmr strains with better persistence in human populations by screening a large population of healthy people, and selecting the dominant LAB strain types for evaluation as probiotics. Conclusion We have shown that specific Lactobacillus strains consumed as part of a feeding study can be tracked through gastrointestinal passage via a colony-based strain typing strategy. The ability to identify specific LAB strains in faeces after human consumption provides a means to answer many important questions concerning the clinical use of probiotics. Our fingerprinting strategy could be used to identify the presence of the LAB isolates of the same genotype as potential probiotics prior to their administration in clinical trials, therefore allowing outcome measures dependent on the

probiotic to be distinguished from those dependent on individuals which may naturally carry the same LAB strain. Overall, the successful application PI3K inhibitor review of molecular epidemiological techniques to cultivable bacterial populations within the human gut provides a platform for future systematic studies on the development of probiotics, as well as a rapid means to assess the strain diversity in healthy versus diseased

humans. Methods Bacterial strains and cultivation Lactobacillus reference strains were obtained from the Belgium Coordinated Collections of Microorganisms (BCCM; http://​bccm.​belspo.​be/​). Additional commercial LAB isolates were obtained from Cultech Ltd (Port Talbot, Wales, UK) or cultured directly from commercially marketed probiotic products as described below; a list of the strains used in this study is shown in Table 2. All strains of LAB were cultivated on MRS agar or in MRS broth (Oxoid, Basingstoke, UK) for 24 to 72 hours at 37°C. Commercial probiotic capsules and powders were resuspended in 5 ml MRS broth MG-132 mw and serial dilutions plated onto MRS agar. To improve the isolation of LAB species from faecal samples, the semi-selective capacity of MRS agar was enhanced by the additional of 120 units per ml of Polymixin B (MRS-P medium; Polymixin B from, Sigma-Aldrich, Gillingham, UK). Fresh growth of purified faecal isolates was swabbed and resuspended in MRS broth containing 8% vol/vol dimethylsulphoxide prior to storage at -80°C. Frozen strains were revived by swabbing the surface of the frozen resuspension and plating onto MRS agar followed by incubation as above.

In addition, it is found that the trilateral structure is an inte

In addition, it is found that the trilateral structure is an interim state in the evolution process from a pristine hexagonal Cilengitide nmr structure to the 5–7 structure. A 5-3-6 structure including this trilateral structure and its adjacent structures would evolve into another 5–7 structure, the right one in Figure  2d, through bond breaking and

bond reforming. Furthermore, a single-chain structure, shown in Figure  2e, can be observed during the fracture process, which can also be found in [26]. Afterwards, the single chain was broken and the indenter totally pierced through the graphene film. Figure 2 Evolution of graphene lattice fracture at different indentation depths. This group of figures shows the process from the state at which the indentation depth reaches

the critical depth to the state the graphene film is totally ruptured with an indenter radius of 2 nm, loading speed of 0.20 Å/ps, and aspect ratio of 1.2. (a) At critical moment: indentation depth 55.95 Å, load 655.08 nN; (b) first broken bond emerged: indentation depth 55.97 Å, load 635.60 nN; (c) pentagonal-heptagonal (5–7) and trilateral structures emerged: indentation depth 55.99 Å, load 426.04 nN; (d) three 5–7 structures: indentation depth 56.01 Å, load 310.45 nN; (e) single-chain structure emerged: indentation depth 56.51 Å, load 112.03 nN; (f) fracture of the chain: indentation depth 56.61 Å, load 93.70 nN. Generally speaking, elastic deformation which is reversible MDV3100 manufacturer and plastic deformation which is irreversible are two

typical kinds of deformation of an object or material in the view of engineering. In order to determine whether the deformation of the graphene film is elastic or plastic, a set of experiments of loading-unloading-reloading processes are conducted. As shown in Figure  3, during the continuous loading process of the indenter on the graphene Dolutegravir supplier film, it can be found that the graphene film mainly takes on two stages in sequence: Figure 3 Load–displacement curve of loading-unloading-reloading process with maximum indentation depth smaller than the critical indentation depth. Stage I. The unloading process is done before the indentation depth reaches the critical depth, d c. The graphene sheet almost can make a complete recovery, i.e., restore its initial structures, and the curves of reloading processes almost perfectly match the initial loading curve while the unloading curve shows very small deviations from the initial one, as shown in the inset of Figure  3. In general, the almost-perfect coincidence is due to the fact that the carbon covalent bonds and the graphene lattice structure are not destroyed. It can be concluded that there is no plastic deformation in this stage, i.e., the graphene undergoes elastic deformation. Stage II, i.e., the yellow region in Figure  3.

The reaction was stopped with PMSF and prepared for immunoblot as

The reaction was stopped with PMSF and prepared for immunoblot as indicated above. Results B. burgdorferi BamA forms multi-protein complexes in the OM Previously, we performed a structural and find more functional characterization of the OM-localized B. burgdorferi BamA protein [32]. Since other BamA orthologs are known to exist in a hetero-oligomeric protein complex [10, 18, 20, 30, 31], we wanted to

determine if native B. burgdorferi BamA could be detected in high molecular weight OM complexes. To perform this assay, we isolated OM vesicles from B. burgdorferi strain B31-A3 and subjected the OM sample to one-dimensional blue native (BN)-PAGE, followed by anti-BamA immunoblot analysis. Results from the immunoblot showed multiple protein bands between the 148 and 1,048 kDa MW markers (Figure 1A), with two prominent bands that resolved at approximately 200 kDa and 1,000 kDa (Figure 1A, arrows). In addition, selleck inhibitor samples from the OM fraction and from the protoplasmic cylinder (PC) fraction were separated by denaturing SDS-PAGE and immunoblotted against

the periplasmic FlaB protein to verify OM purity (Figure 1B). These results demonstrate that native B. burgdorferi BamA is present in multiple high molecular weight OM complexes, which may indicate that BamA associates with other OM-localized proteins or protein complexes. Figure 1 B. burgdorferi BamA is present in OM protein complexes. A. The presence of BamA in OM complexes was revealed by blue native (BN)-PAGE analysis. OM proteins (20 μg) were separated by one-dimensional BN-PAGE (left Olopatadine panel). Subsequently, a strip of BN gel was excised and electrophoretically transferred, and immunoblot analysis was performed with anti-BamA antisera (right panel). Molecular weight standards, in kDa, are indicated at left. Arrows indicate two prominent bands resolving at ~200 kDa and 1000 kDa. B. Purity of a representative OM preparation used for

BN analysis. B. burgdorferi protoplasmic cylinders (PCs) and OMs were isolated by sucrose density gradient centrifugation, as described in Methods. Cell equivalents of OM and PC fractions were separated by SDS-PAGE, electrophoretically transferred onto nitrocellulose membrane, and subsequently immunoblotted with antibodies against BamA and the periplasmic FlaB protein. As expected, BamA is present in the OM, while FlaB is enriched only in the PC fraction. In silico analysis of B. burgdorferi BAM orthologs To identify possible components of the B. burgdorferi BAM complex, our initial approach was to search the B. burgdorferi protein database for putative orthologs of the E. coli BAM lipoproteins, BamB, BamC, BamD, and BamE [18]. Although protein Blast (BlastP) searches using each of the BAM proteins provided no significant sequence matches, BlastP searches using each of the N. meningitidis BAM lipoproteins as a search query yielded one B. burgdorferi protein. This protein, encoded by open reading frame (ORF) bb0324, has significant similarity (P value = 7.2 × 10-5) to the N.

For example, A nidulans bglD (AN7915)

encodes a glucosid

For example, A. nidulans bglD (AN7915)

encodes a glucosidase present in the F9775 biosynthetic gene cluster (Additional file 2). In a cclAΔ strain background in which histone 3 lysine 4 methylation is impaired, the expression of cryptic secondary metabolite clusters, such as F9775, is activated [52]. The activation of bglD expression was observed along with other genes in the F9775 cluster and based on this pattern of coregulation, bglD is included as a member of this cluster [52]. It is unclear, however, whether bglD actually plays a role in F9775 biosynthesis. The gene encoding translation elongation factor 1 gamma, stcT, is a member of the ST gene cluster (stc) of A. nidulans. Its Gamma-secretase inhibitor inclusion in the stc cluster was based on its pattern of coregulation with 24 other genes, some of which have experimentally determined roles in A. nidulans

ST biosynthesis, or are orthologous to A. parasiticus proteins involved selective HDAC inhibitors in AF production, for which ST is a precursor [46]. We also observed a gene, AN2546, that is expressed, and is predicted to encode a glycosylphosphatidylinositol (GPI)-anchored protein [53], located in the emericellamide cluster (Additional file 2); however, an AN2546 deletion strain still produces emericellamide, thus its inclusion in the cluster is based on its genomic location and expression pattern rather than function. These examples indicate that some genes are located within clusters and yet may not contribute to secondary metabolite production. The frequency and significance of unrelated genes that have become incorporated into a secondary metabolism gene cluster remains unclear; experimental verification is needed to further assess these. Ribonuclease T1 In cases where the cluster synteny data were compelling, cluster synteny was given higher precedence than functional annotation in the delineation of the cluster boundaries. Increases in the distance between predicted boundary genes

and the gene directly adjacent to a boundary (which we refer to as intergenic distance) were frequently observed. An example with a large intergenic distance at the right boundary is shown in the A. fumigatus gliotoxin (gli) cluster (Figure 3). However, we found that more subtle increases in intergenic distance were only somewhat reliable when compared to boundaries with experimental evidence. We therefore only based a cluster boundary prediction on an increase in intergenic distance in a small number of cases where no other data were available (Table 9). Discussion AspGD provides high-quality manual and computational gene structure and function annotations for A. nidulans, A. fumigatus, A. niger and A. oryzae, along with sequence analysis and visualization resources for these and additional Aspergilli and related species. Among fungal databases, AspGD is the only resource performing comprehensive manual literature curation for Aspergillus species. AspGD contains curated data covering the entire corpus of experimental literature for A. nidulans, A. fumigatus, A.

3rd edition John Wiley & Sons; 1998 Authors’ contributions JF c

3rd edition. John Wiley & Sons; 1998. Authors’ contributions JF carried out the transcriptional profiling studies and helped to draft the manuscript. LR made measurements of biofilm antibiotic susceptibility and protein synthetic activity. BP assisted with microscopy. FR performed the oxygen microelectrode see more measurements. GE participated in the design of the study and formulation of hypotheses. AP performed the statistical analyses. AM performed the bioinformatic analysis that generated Figure 4. PS conceived the

experimental and analytical approaches, supervised laboratory work and drafted the manuscript. All authors read and approved the final manuscript.”
“Background Most microbes in natural ecosystems exist in highly organized and functional interactive communities, which are composed of cells attached to surfaces and/or to each other either from a single species or multiple species [1–7]. Microbial communities confer a number of advantages for survival, such as nutrient availability with metabolic cooperation, acquisition of new genetic traits, and protection from the environment [4, 8]. The most common microbial communities are biofilms, which refer to assemblages of cell on solid biotic or abiotic surfaces. In recent years, the subject of microbial biofilms has drawn a lot of attention and numerous studies have provided important insights into the genetic basis of biofilm development [5, 7]. Pellicles, arising

from the interface between air and liquid and therefore frequently called air-liquid (A-L) XMU-MP-1 biofilms [9], have been well studied in an array of bacteria, such as Bacillus subtilis, Pseudomonas aeruginosa, and Vibrio parahaemolyticus [7, 10–12]. Pellicle formation consists of at least three distinctive

steps: (i) initial attachment of bacteria to the solid surface (wall of culture nearly device) at the interface between air and liquid, (ii) development of the monolayer pellicle initiated from the attached cells, and (iii) maturation of pellicles with characteristic three-dimensional architecture [1, 11]. In addition to cells, a variety of components, mainly extracellular polymeric substances (EPS), are needed for developing and maintaining the pellicle matrix. The most extensively studied EPS include exopolysaccharides, proteins, and extracellular DNA although contributions of these agents to the integrity of the pellicle matrix may vary [11]. While the pellicle is generally taken into account as a special form of biofilms [5, 7, 13], its distinguishing characteristics justify that this type of biofilm may serve as an independent research model [12–14]. Many factors, including extracellular organelles such as flagella and type IV pili, secreted proteins, and chemical agents supplemented in media such as iron and phosphate, have been shown to play important roles in biofilm formation [5]. However, effects of these factors on the biofilm formation process depend on the bacterium under study.

Many hospitals have created their own unique protocol to address

Many hospitals have created their own unique protocol to address this aspect of management, such as Vanderbilt University Medical Center, which has published their hospital’s guidelines: for the first round of transfusion, 10 units of non-irradiated, uncrossed packed red blood cells, 4 units of AB negative plasma and 2 units of single donor platelets are sent by the blood bank; then for continued hemorrhage, bundles of blood products are sent containing 6 units of non-irradiated PRBCs, 4 units of thawed plasma and 2 units of single donor platelets [18]. in obstetrical patients if transfusion

is needed before type specific C188-9 or crossmatched blood can be obtained, if possible type-O, Rh-negative blood should be utilized because of future risk of Rh sensitization; however if not readily available

Rh-positive blood should not be withheld if clinically required. The surgeon must be aware that hemolytic transfusion reactions with emergency non typed blood can reach up to 5% [19]. Escalated Medical Management If initial interventions fail to control postpartum hemorrhage, https://www.selleckchem.com/products/Belinostat.html a stepwise progression of medical therapy is available using uterotonics to facilitate contraction of the uterus. The first agent used is oxytocin. In the United States, oxytocin is typically administered after delivery of the placenta dosed at 10-20 units in 1000 mL of crystalloid solution, given intravenously (IV) and titrated to an in infusion

rate that achieves adequate uterine contractions. Less commonly, pheromone it can be given intramuscularly (IM) or intrauterine (IU). It is common practice to double the oxytocin in PPH, i.e., 40 units in 1 L, and safety/efficacy has been documented up to 80 units per liter of crystalloid [20]. Oxytocin is not bolused, as boluses can cause hypotension. Excessive oxytocin can cause water intoxication, as it resembles antidiuretic hormone. If there is not adequate uterine tone with oxytocin, the second line agent used will depend on the medications’ side effects and contraindications. Two classes of drugs are available: ergot alkaloids (methylergonovine) or prostaglandins (PGF2α, PGE1, and PGE2). Methylergonovine may be used, dosed as 0.2 mg IM and repeated 2-4 hrs later, as long as the patient does not have hypertension or preeclampsia. If the patient has contraindications to methylergonovine or if the hemorrhage is still non-responsive, 250 μg of 15-methylprostagandin F2α may be injected intramuscularly (IM) up to 3 times at 15-20 minute intervals (maximum dose 2 mg) [21]. Appropriate injection points include thigh, gluteal muscle or directly into the myometrium.