Experimental model of toxin-induced subclinical mastitis and its effect on disruption of follicular function in cows

This study establishes an experimental model for subclinical mastitis induced by Gram-positive (G+) exosecretions of Staphylococcus aureus origin or Gram-negative (G−) endotoxin of Escherichia coli origin to examine its effects on follicular growth and steroid concentrations in Holstein dairy cows. Cows were synchronized with the Ovsynch protocol followed by a series of follicular cycles that included GnRH and PGF_2α doses administered every 8 days. Cows received small intramammary doses of either G+ (10 μg, n = 10) or G− (0.5 μg, n = 6) toxin, or saline (n = 6; uninfected control) every 48 hours for 20 days. Follicular fluids were aspirated from preovulatory follicles before (aspiration one: control), at the end of (aspiration two: immediate effect), and 16 days after the end of (aspiration three: carryover effect) toxin exposure. During the 3 weeks of subclinical mastitis induced by G+ or G−, no local inflammatory signs were detected in the mammary gland and no systemic symptoms were noted: body temperatures of the treated cows did not differ from controls; plasma cortisol and haptoglobin concentrations were not elevated and did not differ among groups. Somatic cell count was higher in the treated groups than in controls, and higher in the G− versus G+ group. For analysis of reproductive responses, cows were further classified as nonaffected or affected based on an more than 20% decline in follicular androstenedione concentration in aspiration two or three relative to the first, control aspiration. Most G− (5/6) and 40% of G+ (4/10) cows were defined as affected by induced mastitis. An immediate decrease in the number of medium-size follicles was recorded on Day 4 of the induced cycle, toward the end of the 20-day mastitis induction, in the affected G+ compared with uninfected control group (1.0 ± 0.5 vs. 3.0 ± 0.4 follicles; P < 0.05); the affected G− and nonaffected G+ subgroups exhibited a similar numerical decline in the number of follicles. A carryover (but not immediate) decrease to 51% and 62% in follicular estradiol concentrations in G− affected group and G+ affected group was detected relative to controls (P < 0.05). The nonaffected G+ subgroup did not differ from its control counterparts. Based on the current experimental model, subclinical IMI induced by G+ or G− toxin disrupts follicular functions, and it seems that the ovarian pool of early antral follicles is susceptible to subclinical mastitis.     

Bactericidal effect of Naja nigricollis toxin γ is related to its membrane-damaging activity

The aim of the present study is to investigate the causal relationship between membrane-damaging activity and bactericidal activity of Naja nigricollis toxin γ. Toxin γ showed a similar inhibitory activity on the growth of Staphylococcus aureus (Gram-positive bacteria) and Escherichia coli (Gram-negative bacteria). Antibacterial activity of toxin γ correlated positively with increase in membrane permeability of bacterial cells. Morphological examination showed that toxin γ disrupted the integrity of bacterial membrane. Toxin γ showed similar binding capability with lipopolysaccharide (LPS) and lipoteichoic acid (LTA), and destabilization of LPS layer and inhibition of LTA biosynthesis on cell wall increased bactericidal effect of toxin γ on E. coli and S. aureus, respectively. Although the potency of toxin γ on permeabilzing model membrane of E. coli and S. aureus was similar, the mode of interaction between toxin γ and model membrane of E. coli and S. aureus differed. Membrane-damaging activity of toxin γ was inhibited by either LPS or LTA. Nevertheless, LPS and LTA altered differently membrane-bound conformation of toxin γ. Taken together, our data suggest that bactericidal activity of toxin γ depends on its ability to induce membrane permeability, and that LPS and LTA structurally suppresses bactericidal effect of toxin γ.     

Antibacterial and membrane‐damaging activities of β‐bungarotoxin B chain

This study investigates whether the B chain of β‐bungarotoxin exerted antibacterial activity against Escherichia coli (Gram‐negative bacteria) and Staphylococcus aureus (Gram‐positive bacteria) via its membrane‐damaging activity. The B chain exhibited a growth inhibition effect on E. coli but did not show a bactericidal effect on S. aureus. The B‐chain bactericidal action on E. coli positively correlated with an increase in membrane permeability in the bacterial cells. Lipopolysaccharide (LPS) layer destabilization and lipoteichoic acid (LTA) biosynthesis inhibition in the cell wall increased the B‐chain bactericidal effect on E. coli and S. aureus. The B chain induced leakage and fusion in E. coli and S. aureus membrane‐mimicking liposomes. Compared with LPS, LTA notably suppressed the membrane‐damaging activity and fusogenicity of the B chain. The B chain showed similar binding affinity with LPS and LTA, whereas LPS and LTA binding differently induced B‐chain conformational change as evidenced by the circular dichroism spectra. Taken together, our data indicate that the antibacterial action of the B chain is related to its ability to induce membrane permeability and suggest that the LPS‐induced and LTA‐induced B‐chain conformational change differently affects the bactericidal action of the B chain. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

The Lipopeptide Antibiotic Paenibacterin Binds to the Bacterial Outer Membrane and Exerts Bactericidal Activity through Cytoplasmic Membrane Damage

Paenibacterin is a broad-spectrum lipopeptide antimicrobial agent produced by Paenibacillus thiaminolyticus OSY-SE. The compound consists of a cyclic 13-residue peptide and an N-terminal C₁₅ fatty acyl chain. The mechanism of action of paenibacterin against Escherichia coli and Staphylococcus aureus was investigated in this study. The cationic lipopeptide paenibacterin showed a strong affinity for the negatively charged lipopolysaccharides (LPS) from the outer membrane of Gram-negative bacteria. Addition of LPS (100 μg/ml) completely eliminated the antimicrobial activity of paenibacterin against E. coli. The electrostatic interaction between paenibacterin and LPS may have displaced the divalent cations on the LPS network and thus facilitated the uptake of antibiotic into Gram-negative cells. Paenibacterin also damaged the bacterial cytoplasmic membrane, as evidenced by the depolarization of membrane potential and leakage of intracellular potassium ions from cells of E. coli and S. aureus. Therefore, the bactericidal activity of paenibacterin is attributed to disruption of the outer membrane of Gram-negative bacteria and damage of the cytoplasmic membrane of both Gram-negative and Gram-positive bacteria. Despite the evidence of membrane damage, this study does not rule out additional bactericidal mechanisms potentially exerted by paenibacterin.     

Antimicrobial activity of highly stable silver nanoparticles embedded in agar-agar matrix as a thin film

Highly stable silver nanoparticles (Ag NPs) in agar-agar (Ag/agar) as inorganic-organic hybrid were obtained as free-standing film by in situ reduction of silver nitrate by ethanol. The antimicrobial activity of Ag/agar film on Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), and Candida albicans (C. albicans) was evaluated in a nutrient broth and also in saline solution. In particular, films were repeatedly tested for antimicrobial activity after recycling. UV-vis absorption and TEM studies were carried out on films at different stages and morphological studies on microbes were carried out by SEM. Results showed spherical Ag NPs of size 15-25 nm, having sharp surface plasmon resonance (SPR) band. The antimicrobial activity of Ag/agar film was found to be in the order, C. albicans > E. coli > S. aureus, and antimicrobial activity against C. albicans was almost maintained even after the third cycle. Whereas, in case of E. coli and S. aureus there was a sharp decline in antimicrobial activity after the second cycle. Agglomeration of Ag NPs in Ag/agar film on exposure to microbes was observed by TEM studies. Cytotoxic experiments carried out on HeLa cells showed a threshold Ag NPs concentration of 60 μg/mL, much higher than the minimum inhibition concentration of Ag NPs (25.8 μg/mL) for E. coli. The mechanical strength of the film determined by nanoindentation technique showed almost retention of the strength even after repeated cycle.     

Use of Hydrostatic Pressure for Inactivation of Microbial Contaminants in Cheese

The objective of this study was to determine the effect of high pressure (HP) on the inactivation of microbial contaminants in Cheddar cheese (Escherichia coli K-12, Staphylococcus aureus ATCC 6538, and Penicillium roqueforti IMI 297987). Initially, cheese slurries inoculated with E. coli, S. aureus, and P. roqueforti were used as a convenient means to define the effects of a range of pressures and temperatures on the viability of these microorganisms. Cheese slurries were subjected to pressures of 50 to 800 MPa for 20 min at temperatures of 10, 20, and 30°C. At 400 MPa, the viability of P. roqueforti in cheese slurry decreased by >2-log-unit cycles at 10°C and by 6-log-unit cycles at temperatures of 20 and 30°C. S. aureus and E. coli were not detected after HP treatments in cheese slurry of >600 MPa at 20°C and >400 MPa at 30°C, respectively. In addition to cell death, the presence of sublethally injured cells in HP-treated slurries was demonstrated by differential plating using nonselective agar incorporating salt or glucose. Kinetic experiments of HP inactivation demonstrated that increasing the pressure from 300 to 400 MPa resulted in a higher degree of inactivation than increasing the pressurization time from 0 to 60 min, indicating a greater antimicrobial impact of pressure. Selected conditions were subsequently tested on Cheddar cheese by adding the isolates to cheese milk and pressure treating the resultant cheeses at 100 to 500 MPa for 20 min at 20°C. The relative sensitivities of the isolates to HP in Cheddar cheese were similar to those observed in the cheese slurry, i.e., P. roqueforti was more sensitive than E. coli, which was more sensitive than S. aureus. The organisms were more sensitive to pressure in cheese than slurry, especially with E. coli. On comparison of the sensitivities of the microorganisms in a pH 5.3 phosphate buffer, cheese slurry, and Cheddar cheese, greatest sensitivity to HP was shown in the pH 5.3 phosphate buffer by S. aureus and P. roqueforti while greatest sensitivity to HP by E. coli was exhibited in Cheddar cheese. Therefore, the medium in which the microorganisms are treated is an important determinant of the level of inactivation observed.     

Non-Classical ProIL-1beta Activation during Mammary Gland Infection Is Pathogen-Dependent but Caspase-1 Independent

Infection of the mammary gland with live bacteria elicits a pathogen-specific host inflammatory response. To study these host-pathogen interactions wild type mice, NF-kappaB reporter mice as well as caspase-1 and IL-1beta knockout mice were intramammarily challenged with Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The murine mastitis model allowed to compare the kinetics of the induced cytokine protein profiles and their underlying pathways. In vivo and ex vivo imaging showed that E. coli rapidly induced NF-kappaB inflammatory signaling concomitant with high mammary levels of TNF-alpha, IL-1 alpha and MCP-1 as determined by multiplex analysis. In contrast, an equal number of S. aureus bacteria induced a low NF-kappaB activity concomitant with high mammary levels of the classical IL-1beta fragment. These quantitative and qualitative differences in local inflammatory mediators resulted in an earlier neutrophil influx and in a more extensive alveolar damage post-infection with E. coli compared to S. aureus. Western blot analysis revealed that the inactive proIL-1beta precursor was processed into pathogen-specific IL-1beta fragmentation patterns as confirmed with IL-1beta knockout animals. Additionally, caspase-1 knockout animals allowed to investigate whether IL-1beta maturation depended on the conventional inflammasome pathway. The lack of caspase-1 did not prevent extensive proIL-1beta fragmentation by either of S. aureus or E. coli. These non-classical IL-1beta patterns were likely caused by different proteases and suggest a sentinel function of IL-1beta during mammary gland infection. Thus, a key signaling nodule can be defined in the differential host innate immune defense upon E. coli versus S. aureus mammary gland infection, which is independent of caspase-1.     

Characterization and bioactivity of hepcidin-2 in zebrafish: Dependence of antibacterial activity upon disulfide bridges

Hepcidin is an antimicrobial peptide and iron-regulatory molecule with highly conserved disulfide bridges among vertebrates, but structural insights into the function in fish remains largely missing. We demonstrate here that recombinant hepcidin-2 from zebrafish is capable of inhibiting the growth of the Gram-negative bacteria Escherichia coli and Vibrio anguillarum, and the Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis with minimum inhibitory concentrations (MICs) of 18, 15, 13 and 9 μM, respectively. We also show by TEM examination that recombinant hepcidin-2 is directly cidal to the cells of E. coli and S. aureus. Moreover, we find that hepcidin-2 displays affinity to LPS, LTA and PGN. All these data indicate that hepcidin-2 is both a pattern recognition molecule, capable of identifying LPS, LTA and PGN, and an antibacterial effector, capable of inhibiting the growth of bacteria. The data also show that the antibacterial activity of hepcidin-2 depends upon the disulfide bridges.     

Purification and biochemical characterization of Mur ligases from Staphylococcus aureus

The Mur ligases (MurC, MurD, MurE and MurF) catalyze the stepwise synthesis of the UDP-N-acetylmuramoyl-pentapeptide precursor of peptidoglycan. The murC, murD, murE and murF genes from Staphylococcus aureus, a major pathogen, were cloned and the corresponding proteins were overproduced in Escherichia coli and purified as His₆-tagged forms. Their biochemical properties were investigated and compared to those of the E. coli enzymes. Staphylococcal MurC accepted l-Ala, l-Ser and Gly as substrates, as the E. coli enzyme does, with a strong preference for l-Ala. S. aureus MurE was very specific for l-lysine and in particular did not accept meso-diaminopimelic acid as a substrate. This mirrors the E. coli MurE specificity, for which meso-diaminopimelic acid is the preferred substrate and l-lysine a very poor one. S. aureus MurF appeared less specific and accepted both forms (l-lysine and meso-diaminopimelic acid) of UDP-MurNAc-tripeptide, as the E. coli MurF does. The inverse and strict substrate specificities of the two MurE orthologues is thus responsible for the presence of exclusively meso-diaminopimelic acid and l-lysine at the third position of the peptide in the peptidoglycans of E. coli and S. aureus, respectively. The specific activities of the four Mur ligases were also determined in crude extracts of S. aureus and compared to cell requirements for peptidoglycan biosynthesis.     

Identification of Staphylococcus aureus and Escherichia coli isolated from Egyptian food by conventional and molecular methods

Staphylococcus aureus and Escherichia coli were enumerated and isolated from ready-to-eat vegetables salad and meat luncheon on their selective media (Baird-parker and Macconkey agar, respectively). Twenty suspicious colonies of each (10 from each product) were randomly chosen and identified using conventional based on morphological and physiological characteristics. S. aureus and E. coli isolates which gave the highest pathogenicity were chosen for identification and confirmation with molecular method based on 16S rRNA gene. The PCR amplification method of 16S rRNA gave the same identification results as conventional method, but it was sensitive and fast. This molecular method takes about 48 h in comparison with 6 days for conventional method. The 16S rRNA of S. aureus and E. coli were deposited in the Genebank database under accessions (AB599719.1 and AB599716.1, respectively).     

Modification of membrane properties and fatty acids biosynthesis-related genes in Escherichia coli and Staphylococcus aureus: Implications for the antibacterial mechanism of naringenin

In this work, modifications of cell membrane fluidity, fatty acid composition and fatty acid biosynthesis-associated genes of Escherichia coli ATCC 25922 (E. coli) and Staphylococcus aureus ATCC 6538 (S. aureus), during growth in the presence of naringenin (NAR), one of the natural antibacterial components in citrus plants, was investigated. Compared to E. coli, the growth of S. aureus was significantly inhibited by NAR in low concentrations. Combination of gas chromatography–mass spectrometry with fluorescence polarization analysis revealed that E. coli and S. aureus cells increased membrane fluidity by altering the composition of membrane fatty acids after exposure to NAR. For example, E. coli cells produced more unsaturated fatty acids (from 18.5% to 43.3%) at the expense of both cyclopropane and saturated fatty acids after growth in the concentrations of NAR from 0 to 2.20 mM. For S. aureus grown with NAR at 0 to 1.47 mM, the relative proportions of anteiso-branched chain fatty acids increased from 37.2% to 54.4%, whereas iso-branched and straight chain fatty acids decreased from 30.0% and 33.1% to 21.6% and 23.7%, respectively. Real time q-PCR analysis showed that NAR at higher concentrations induced a significant down-regulation of fatty acid biosynthesis-associated genes in the bacteria, with the exception of an increased expression of fabA gene. The minimum inhibitory concentration (MIC) of NAR against these two bacteria was determined, and both of bacteria underwent morphological changes after exposure to 1.0 and 2.0 MIC.     

Serine-based surfactants as effective antimicrobial agents against multiresistant bacteria

The antimicrobial activity of two serine derived gemini cationic surfactants, amide (12Ser)₂CON12 and ester (12Ser)₂COO12, was tested using sensitive, E. coli ATCC 25922 and S. aureus ATCC 6538, and resistant, E. coli CTX M2, E. coli TEM CTX M9 and S. aureus ATCC 6538 and S. aureus MRSA ATCC 43300 Gram-positive and Gram-negative bacteria strains. Very low MIC values (5 μM) were found for the two resistant strains E.coli TEM CTX M9 and S. aureus MRSA ATCC 43300, in the case of the amide derivative, and for S. aureus MRSA ATCC 43300, in the case of the ester derivative. The interaction of the serine amphiphiles with lipid-model membranes (DPPG and DPPC) was investigated using Langmuir monolayers. A more pronounced effect on the DPPG than on the DPPC monolayer was observed. The effect induced by the surfactants on bacteria membrane was explored by Atomic Force Microscopy. A clear disruption of the bacteria membrane was observed for E. coli TEM CTX M9 upon treatment with (12ser)₂CON12, whereas for the S. aureus MRSA few observable changes in cell morphology were found after treatment with either of the two surfactants. The cytotoxicity of the two compounds was assessed by hemolysis assay on human red blood cells (RBC). The compounds were shown to be non-cytotoxic up to 10 μM. Overall, the results reveal a promising potential, in particular of the amide derivative, as antimicrobial agent for two strains of antibiotic resistant bacteria.     

Bacteria May Cope Differently from Similar Membrane Damage Caused by the Australian Tree Frog Antimicrobial Peptide Maculatin 1.1

Maculatin 1.1 (Mac1) is an antimicrobial peptide from the skin of Australian tree frogs and is known to possess selectivity toward Gram-positive bacteria. Although Mac1 has membrane disrupting activity, it is not known how Mac1 selectively targets Gram-positive over Gram-negative bacteria. The interaction of Mac1 with Escherichia coli, Staphylococcus aureus, and human red blood cells (hRBC) and with their mimetic model membranes is here reported. The peptide showed a 16-fold greater growth inhibition activity against S. aureus (4 μm) than against E. coli (64 μm) and an intermediate cytotoxicity against hRBC (30 μm). Surprisingly, Sytox Green uptake monitored by flow cytometry showed that Mac1 compromised both bacterial membranes with similar efficiency at ∼20-fold lower concentration than the reported minimum inhibition concentration against S. aureus. Mac1 also reduced the negative potential of S. aureus and E. coli membrane with similar efficacy. Furthermore, liposomes mimicking the cell membrane of S. aureus (POPG/TOCL) and E. coli (POPE/POPG) were lysed at similar concentrations, whereas hRBC-like vesicles (POPC/SM/Chol) remained mostly intact in the presence of Mac1. Remarkably, when POPG/TOCL and POPE/POPG liposomes were co-incubated, Mac1 did not induce leakage from POPE/POPG liposomes, suggesting a preference toward POPG/TOCL membranes that was supported by surface plasma resonance assays. Interestingly, circular dichroism spectroscopy showed a similar helical conformation in the presence of the anionic liposomes but not the hRBC mimics. Overall, the study showed that Mac1 disrupts bacterial membranes in a similar fashion before cell death events and would preferentially target S. aureus over E. coli or hRBC membranes.     

Antimicrobial activity of quinoxaline 1,4-dioxide with 2- and 3-substituted derivatives

Quinoxaline is a chemical compound that presents a structure that is similar to quinolone antibiotics. The present work reports the study of the antimicrobial activity of quinoxaline N,N-dioxide and some derivatives against bacterial and yeast strains. The compounds studied were quinoxaline-1,4-dioxide (QNX), 2-methylquinoxaline-1,4-dioxide (2MQNX), 2-methyl-3-benzoylquinoxaline-1,4-dioxide (2M3BenzoylQNX), 2-methyl-3-benzylquinoxaline-1,4-dioxide (2M3BQNX), 2-amino-3-cyanoquinoxaline-1,4-dioxide (2A3CQNX), 3-methyl-2-quinoxalinecarboxamide-1,4-dioxide (3M2QNXC), 2-hydroxyphenazine-N,N-dioxide (2HF) and 3-methyl-N-(2-methylphenyl)quinoxalinecarboxamide-1,4-dioxide (3MN(2MF)QNXC). The prokaryotic strains used were Staphylococcus aureus ATCC 6538, S. aureus ATCC 6538P, S. aureus ATCC 29213, Escherichia coli ATCC 25922, E. coli S3R9, E. coli S3R22, E. coli TEM-1 CTX-M9, E. coli TEM-1, E. coli AmpC Mox-2, E. coli CTX-M2 e E. coli CTX-M9. The Candida albicans ATCC 10231 and Saccharomyces cerevisiae PYCC 4072 were used as eukaryotic strains. For the compounds that presented activity using the disk diffusion method, the minimum inhibitory concentration (MIC) was determined. The alterations of cellular viability were evaluated in a time-course assay. Death curves for bacteria and growth curves for S. cerevisiae PYCC 4072 were also accessed. The results obtained suggest potential new drugs for antimicrobial activity chemotherapy since the MIC's determined present low values and cellular viability tests show the complete elimination of the bacterial strain. Also, the cellular viability tests for the eukaryotic model, S. cerevisiae, indicate low toxicity for the compounds tested.     

The influence of polyhexamethylene guanidine derivatives introduced into polyhydroxybutyrate on biofilm formation and the activity of bacterial enzymes

Escherichia coli and Staphylococcus aureus were able to produce biofilm on the surface of polyhydroxybutyrate (PHB), but their abundance depended on type and the concentrations of the polyhexamethylene guanidine (PHMG) derivatives introduced in PHB. Different types of PHMG derivatives inhibited S. aureus ATCC 6538P biofilm formation, but PHB with PHMG salt of sulfanilic acid stimulated E. coli ATCC 8739 biofilm formation. The presence of all PHMG derivatives decreased significantly the number of viable cells of the test bacteria directly proportional to the concentration of the biocidal agent. PHMG derivatives affected the activity of microbiological hydrolases with different degrees. Some of them (PHB with PHMG stearate) stimulated activity of E. coli ATCC 8739 hydrolases, other (PHB with the PHMG salt of sulfanilic acid) inhibited activity of the S. aureus ATCC 6538P hydrolases. The PHMG derivatives introduced in PHB also inhibited the activity of bacterial dehydrogenases.     

The Role of O-antigen in P1 Transduction of Shigella flexneri and Escherichia coli with its Alternative S' Tail Fibre

Enterobacteria phage P1 expresses two types of tail fibre, S and S'. Despite the wide usage of phage P1 for transduction, the host range and the receptor for its alternative S' tail fibre was never determined. Here, a ΔS-cin Δpac E. coli P1 lysogenic strain was generated to allow packaging of phagemid DNA into P1 phage having either S or S' tail fibre. P1(S') could transduce phagemid DNA into Shigella flexneri 2a 2457O, Shigella flexneri 5a M90T and Escherichia coli O3 efficiently. Mutational analysis of the O-antigen assembly genes and LPS inhibition assays indicated that P1(S') transduction requires at least one O-antigen unit. E. coli O111:B4 LPS produced a high neutralising effect against P1(S') transduction, indicating that this E. coli strain could be susceptible to P1(S')-mediated transduction. Mutations in the O-antigen modification genes of S. flexneri 2a 2457O and S. flexneri 5a M90T did not cause significant changes to P1(S’) transduction efficiency. A higher transduction efficiency of P1(S') improved the delivery of a cas9 antimicrobial phagemid into both S. flexneri 2457O and M90T. These findings provide novel insights into P1 tropism-switching, by identifying the bacterial strains which are susceptible to P1(S')-mediated transduction, as well as demonstrating its potential for delivering a DNA sequence-specific Cas9 antimicrobial into clinically relevant S. flexneri.     

Gene Expression-Based Classifiers Identify Staphylococcus aureus Infection in Mice and Humans

Staphylococcus aureus causes a spectrum of human infection. Diagnostic delays and uncertainty lead to treatment delays and inappropriate antibiotic use. A growing literature suggests the host’s inflammatory response to the pathogen represents a potential tool to improve upon current diagnostics. The hypothesis of this study is that the host responds differently to S. aureus than to E. coli infection in a quantifiable way, providing a new diagnostic avenue. This study uses Bayesian sparse factor modeling and penalized binary regression to define peripheral blood gene-expression classifiers of murine and human S. aureus infection. The murine-derived classifier distinguished S. aureus infection from healthy controls and Escherichia coli-infected mice across a range of conditions (mouse and bacterial strain, time post infection) and was validated in outbred mice (AUC>0.97). A S. aureus classifier derived from a cohort of 94 human subjects distinguished S. aureus blood stream infection (BSI) from healthy subjects (AUC 0.99) and E. coli BSI (AUC 0.84). Murine and human responses to S. aureus infection share common biological pathways, allowing the murine model to classify S. aureus BSI in humans (AUC 0.84). Both murine and human S. aureus classifiers were validated in an independent human cohort (AUC 0.95 and 0.92, respectively). The approach described here lends insight into the conserved and disparate pathways utilized by mice and humans in response to these infections. Furthermore, this study advances our understanding of S. aureus infection; the host response to it; and identifies new diagnostic and therapeutic avenues.     

Follicular atresia and LH concentrations during the follicular phase of the estrous cycle in the goat (Capra hircus)

The objective of the study was to identify the effects of LH on the final follicle maturation process as well as the incidence of atresia during the follicular phase of the goat's estrous cycle. In Experiment 1, concentrations of the LH were measured during the follicular phase of a synchronized cycle in 8 Canary goats. In Experiment 2, the same animals were synchronized again. On each day of a 4-day experimental period (day 0=day of sponges withdrawal), 2 of the goats were bilaterally ovariectomized. Follicles with a diameter >1 mm were dissected out to obtain qualitative histological data in normal, early atretic I, early atretic II, advanced atretic I and advanced atretic II follicles. The total interval from sponge withdrawal to LH peak was 77.5±9.8 h. LH peak concentration averaged 44±5.3 ng/ml and the mean length of the preovulatory surge (amounts over 10 ng/ml) was 8.9±0.9 h. During the total follicular phase, there were more atretic follicles than normal follicles (58 vs. 30, P<0.05). The number of early and advanced atretic follicles was similar. There were more early atresia I than early atresia II follicles (23 vs. 6, P<0.05). On day 2, the number of advanced atretic follicles was greater than early atretic follicles (10 vs. 4, P<0.05). There was an increase in the number of early atretic follicles from day 2 to day 4 (4 vs. 9, P<0.05), which was consistent with the effects of the preovulatory LH surge.     

In Vitro Antimicrobial Activity and Human Pharmacology of Cephalexin, a New Orally Absorbed Cephalosporin C Antibiotic

Concentrations of cephalexin (an orally absorbed derivative of cephalosporin C) in serum and urine were determined in normal volunteers and patients. The in vitro antibacterial activity was also studied. All strains of group A β-hemolytic streptococci and Diplococcus pneumoniae were inhibited by 3.1 μg/ml. Of the Staphylococcus aureus strains, 88% were inhibited by 6.3 μg/ml, and 12.5 μg/ml was inhibitory for all S. aureus, 80% of Escherichia coli, 72% of Klebsiella-Aerobacter, and 56% of Proteus mirabilis strains. About 90 to 96% of E. coli, Klebsiella Aerobacter, and P. mirabilis strains were inhibited by 25 μg of cephalexin per ml. Pseudomonas and indole-positive Proteus strains proved to be quite resistant to cephalexin. Cephalexin was well absorbed after oral administration. A peak serum concentration of cephalexin of at least 5 μg/ml was achieved in each volunteer with 250 and 500-mg doses. A mean peak serum concentration of 7.7 μg/ml was achieved with 250-mg doses; 12.3μg/ml was achieved with 500-mg doses of antibiotic. Food did not interfere with absorption. Probenecid enhanced both the peak serum concentration and the duration of antibiotic activity in the serum. Over 90% of the administered dose was excreted in the urine within 6 hr. The mean peak serum concentration of cephalexin after an oral dose of 500 mg was adequate to inhibit all group A streptococci, D. pneumoniae, and S. aureus, 85% of E. coli, and about 40 to 75% of Klebsiella-Aerobacter and P. mirabilis strains. Levels of cephalexin in urine were adequate to inhibit over 90% of E. coli, and P. mirabilis and 80 to 96% of Klebsiella-Aerobacter strains.