A pele humana é o maior e um dos mais importantes órgãos, esta é a principal barreira entre o corpo e o meio externo, sempre que há um desequilíbrio no mecanismo de defesa inata, o risco de uma infecção é proeminente. A queimadura é um quadro clínico em que a barreira epitelial é danificada, resultado em uma defesa imunológica deficitária e uma grande abundância de nutrientes, o que favorece a proliferação de micro-organismos. No presente estudo foi desenvolvido e caracterizado uma suspensão inovadora de nanocápsulas poliméricas de poli(ε-caprolactona) com triclosan e α-bisabol, revestida com quitosana (NCCQ) para aplicação tópica em feridas de queimadura, com intuito de reduzir a contaminação microbiana. Foram realizados ensaios de caracterização físico-química, microbiológica e cutânea. Os resultados obtidos para os ensaios de caracterização mostraram que a suspensão de nanocápsulas apresentou diâmetro de partícula adequado, ausência de cristais ou contaminação micrométrica, potencial zeta catiônico, pH levemente ácido, com uma alta eficiência de encapsulação e estável no período de trinta dias. Os ensaios microbiológicos foram realizados frente Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa e Candida albicans, através de microdiluição seriada e teste do desafio. O primeiro ensaio mostrou uma redução no valor da concentração inibitória mínima e melhores resultados para quase todas as cepas analisadas frente aos controles e dados da literatura, inclusive P. aeruginosa, a qual possui uma alta resistência ao triclosan. O teste do desafio serviu para comprovar que o efeito antimicrobiano da suspensão, se manteve ao longo de 28 dias, mesmo quando incorporada ao curativo de hemi-celulose. Os ensaios cutâneos de permeação/penetração e lavabilidade mostraram que a suspensão de nanocápsulas NCCQ teve uma retenção no estrato córneo, o que é desejável para uma formulação tópica. / The human skin is the largest and one of the most important organs, this is the main barrier between the body and the external environment, whenever there is an imbalance in innate defense mechanism, the risk of infection is prominent. Burn is a clinical condition in which the epithelial barrier is damaged, resulting in a deficient immune defense, also the abundance of nutrients favors to proliferation of microorganisms. In the present study it was developed and characterized a novel suspension of polymeric nanocapsules of poly (ε-caprolactone) with triclosan and α-bisabol coated with chitosan (NCCQ) for topical application in burn wounds, in order to reduce microbial contamination. Physical-chemical, microbiological and skin tests were performed in order to prove its applicability. The results obtained for the characterization tests showed that the suspension of nanocapsules had suitable particle diameter, no crystals or micrometric contamination cationic zeta potential, slightly acid pH with a high encapsulation efficiency and stable within thirty days. Microbiological tests were carried forward Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans, via serial microdilution and challenge test. The first experiment showed a reduction in the value of the minimum inhibitory concentration and best results for nearly all strains analyzed when compared with controls and data from the literature, including P. aeruginosa, which has a high resistance to triclosan. The challenge test was used to demonstrate that the antimicrobial effect of the suspension was maintained over 28 days even when incorporated into the dressing hemi-cellulose. The skin permeation/penetration and washability tests of NCCQ nanocapsule suspension showed that it had retention in the stratum corneum, which is desirable for a topical formulation.

Agentes com propriedades antimicrobianas e antifúngicas são nanoencapsuladas em nanopartículas tendo em vista aumentar os efeitos contra as infecções microbiológicas causadas por Aspergillus ssp. e Staphylococcus aureus. Dentre os sistemas veiculares para entrega de fármacos as nanocápsulas de núcleo lipídico se destacam e tem sido largamente estudadas devido aos seus potenciais terapêuticos. Neste estudo desenvolvemos e caracterizamos as nanocápsulas de núcleo lipídico revestidas com quitosana para assim associar efeitos antimicrobianos, do fármaco e de material de revestimento das nanocápsulas. Para tanto, encapsulamos a dapsona nas nanocápsulas com o propósito obter sua dupla atividade antimicrobiana, associada à da quitosana, material de revestimento. Posteriormente, as nanocápsulas contendo dapsona e revestidas com quitosana foram secas pela técnica de spray-drying a fim de desenvolver uma plataforma tecnológica para a obtenção de produtos intermediários e finais, para futuras aplicações pulmonares ou tópicas dos pós de nanocápsulas. As formulações de nanocápsulas de núcleo lipídico e as com dapsona revestidas com fosfatidilcolina e polissorbato 80 e fosfatidilcolina, polissorbato 80 e quitosana apresentaram distribuição unimodal. Por meio da microscopia eletrônica de transmissão foi possível observar a coroa hidrofilica das nanocápsulas constituida de estruturas micelas. Os resultados demonstraram uma redução de até dez vezes na concentração de dapsona quando nanoencapsulada em sistema nanoestruturado revestidos com fosfatidilcolina, polissorbato 80 e quitosana em comparação com a administração da dapsona em solução para efeitos antibacterianos. As formulações de nanocápsulas de núcleo lipídico sem o fármaco revestidas com fosfatidilcolina, polissorbato 80 e quitosana também inibiram o crescimento bacteriano e fúngicos quando a quitosana foi administrada nas concentrações relativas. O efeito da quitosana demostrou elevado potencial para inibir o crescimento bacteriano e fúngico. Os pós das nanocápsulas contendo dapsona revestidas com quitosana e secas com leucina apresentaram produtos intermediários para futuras aplicações biológicas, tanto para via respiratória quanto para via tópica a fim coibir as infecções causadas pelos microorganismos. / Agents with antimicrobial and antifungal properties were encapsulated into nanoparticles in order to improve the effects against microbial infections caused by Aspergillus spp. and Staphylococcus aureus. Among the carriers for drug delivery systems of the lipid core nanocapsules have been widely studied because of their therapeutic potential. In this study we have developed and characterized the lipid core nanocapsules coated with chitosan thus to associate antimicrobial effects of the drug to nanocapsules of the coating material. To this end, dapsone was encapsulated into the nanocapsules in order to obtain a double antimicrobial activity, associated to the chitosan coating material. Subsequently, the nanocapsules containing dapsone and coated with chitosan were dried by spray-drying technique in order to develop a technological platform to obtain intermediates and final products for future pulmonary or topical application of nanocapsules powders. The lipid core nanocapsules with and without dapsone coat with phosphatidylcholine and phosphatidyl choline and polysorbate 80, polysorbate 80, and chitosan showed unimodal distribution. By transmission electron microscopy it was possible to observe the crown of hydrophilic nanocapsules consisting of micellar structures. The results showed a reduction of up to ten times the concentration of dapsone when nanocoated in nanostructured system coated with phosphatidylcholine, polysorbate 80 and chitosan compared to administration of dapsone in solution for antibacterial effects.The lipid core nanocapsule formulations without drug coated with phosphatidylcholine, polysorbate 80, chitosan also inhibit bacterial and fungal growth when chitosan was administered in relative concentrations. The effect of chitosan demonstrated high potential to inhibit bacterial and fungal growth.The powder of nanocapsules containing dapsone coated with chitosan and dried with leucine showed intermediate products for future biological applications, for both the airway and topically to prevent infections caused by microorganisms.

>Magister Scientiae - MSc / This thesis reports on the syntheses of complexes of (arene) ruthenium (II) isoniazid Schiff base ligands for antimicrobial studies. Isoniazid Schiff base ligands; isonicotinyl acid (2-hydroxy-5- methyl-benzilidene)-hydrazide (L1), isonicotinyl acid (2-hydroxy-5-methoxy-benzilidene)- hydrazide (L2), isonicotinyl acid (-5-chloro-2-hydroxy-benzilidene)-hydrazide (L3), isonicotinyl acid (-5-bromo-2-hydroxy-benzilidene)-hydrazide (L4), isonicotinyl acid (2-hydroxy-5-nitrobenzilidene)- hydrazide (L5) were prepared by condensation reaction under reflux from equimolar amounts of isioniazid, which is an amine, with five different aldehyde moieties. Ruthenium (II) complexes of these isoniazid Schiff base ligands (C1 - C5) were prepared in an ethanolic solution under reflux and inert atmosphere at 60°C using Schlenk techniques. Fourier transform infrared spectroscopy (FTIR), ultraviolet – visible spectroscopy, thermogravimetric analysis, nuclear magnetic resonance and elemental analysis were the characterization techniques that confirmed the successful preparation of the ligands. All the ligands spectra displayed the imine functional group peak which confirmed the successful preparation. The ligands L1 – L5 and the complexes C1 – C5 were subjected to similar characterization techniques which further confirmed the successful syntheses and the coordination of metal and ligand by displaying a shift in their respective imine peaks and transitions values. All the synthesized compounds were subjected to a standard antimicrobial test using three microorganisms, Staphylococcus aureus, Methicillin resistant Staphylococcus aureus and Pseudomonas aeruginosa. Out the ten compounds tested, only ligand L5 gave the best results against Staphylococcus aureus.

Cystic fibrosis (CF) is a hereditary multi-organ disorder characterized by formation of thick, viscous mucus in the lungs, leading to decreased fluid clearance and significant bacterial colonization. The bacteria form colonies, called biofilms, that are attached to the mucosal surface and produce a protective polymeric matrix. The matrix helps the biofilms form stable structures in the lungs while also protecting the embedded bacterial colonies from the host defense system and antimicrobials. Pseudomonas aeruginosa are opportunistic bacteria that commonly infect CF airways in the biofilm form. Current antibiotic treatment regimens fail to completely eradicate these biofilms, leading to chronic, persistent infections that over time lead to patient death. Therefore, there is a need to investigate antibacterial strategies that would completely eradicate these infections at reasonable doses and improve quality of patients’ lives. In this thesis, two strategies are investigated to better eradicate bacterial colonies – (1) the use of nutrient dispersion compounds for increasing the susceptibility of biofilm bacteria to the co-administered antibiotics, and (2) PEGylation of antimicrobial peptides to increase peptide retention in the lung airways. Clinical strains of P. aeruginosa isolated from lungs of CF patients were used in this research to better mimic the greater robustness of clinical biofilms compared to biofilms of laboratory bacterial strains. Growth curve studies were carried out to characterize the growth patterns of the bacterial strains. Antibiotic susceptibility of the planktonic (free-flowing) bacteria was studied using the minimum inhibitory concentration (MIC) assay. A method to grow and characterize 1-day and 4-day old biofilms in the minimum biofilm eradication concentration (MBEC) assay apparatus was developed and characterized. The MBECs of combination formulations consisting of an antibiotic and a nutrient dispersion compound for different treatment durations were measured against biofilms of the clinical isolates using four commonly used antibiotics, and sodium citrate as the nutrient dispersion compound. The growth curve studies allowed for better understanding of the clinical isolates’ growth rates in vitro, which could play an important role on their susceptibility to antibiotics. All bacterial strains displayed susceptibility to tobramycin sulfate and ciprofloxacin hydrochloride. Uniform bacterial growth was observed for 1-day old biofilms of both clinical isolates across all pegs. Growing 4-day old biofilms using 100% MHB without refreshing the bacterial suspension over 4 days gave uniform biofilm bacterial growth across the pegs. Four-day old biofilms displayed greater biomass than 1-day old biofilms for 2 out of 3 bacterial strains. Combination formulations eradicated 1-day and 4-day old biofilms at lower antibiotic concentrations than the antibiotic alone, with further improvement in eradication after increasing the duration of treatment. Sodium citrate did not enhance the metabolic activity of the biofilm bacteria. The antimicrobial peptide CaLL was conjugated with different MW polyethylene glycol (PEG) molecules using disulfide and maleimide linkages, and the effect of PEGylation on its antibacterial activity against P. aeruginosa laboratory strain PAO1 was evaluated. PEGylation was observed to reduce bacterial growth inhibition by CaLL, with the disulfide-linked CaLL-PEG less efficacious than the maleimide-linked CaLL-PEG. Time-kill assays demonstrated the longer duration of action of PEGylated peptides compared to non-PEGylated peptides, probably due to prevention of enzymatic degradation of the peptide by the PEG molecule. This research will shed light on antibacterial strategies for complete and rapid eradication of bacterial biofilms, thereby reducing development of antibiotic resistance and prevent recurrence of infection, reducing progressive lung damage caused in people with CF, and improve their quality of life.

Cationic antimicrobial peptides (CAPs) are an important component of the innate immune system and are instrumental in the elimination of bacteria, viruses, protozoa, yeast, fungi and cancerous cells from the body. CAPs are comprised of less than 100 amino acids and have a net positive charge due to a multitude of basic residues in their primary sequences. CAPs exert their antimicrobial activity primarily through the formation of pores in microbial membranes, but also play important immunostimulatory roles in the body. Glycosaminoglycans (GAGs) are negatively charged, polydisperse linear polysaccharides found at cellular surfaces. Although many protein-binding interactions of the GAG family, including heparin and heparan sulfate, have been well-characterized, it is not known to what extent endogenous GAGs affect the innate immune system. In the studies here the modulatory activities of GAGs and other polyanionic polysaccharides (PPSs) on CAPs were probed. Initial studies focused on interactions between a short peptide derived from bovine lactoferricin and GAGs. GAGs and other PPSs were then tested for their ability to modulate the antimicrobial activities of a number of CAPs against Gram-positive and -negative organisms. GAGs were also tested for the ability to modulate CAPs binding to bacterial lipopolysaccharide. CAP affinities for the GAGs were determined from lipopolysaccharide competition binding assays. Finally GAGs were evaluated for the ability to protect CAPs from proteolytic degradation. The modulatory activities of GAGs and other PPSs are largely dependent upon all components of the test system and, to a lesser extent, the charge of the molecule.

Fifty three Staphylococcus aureus isolates were obtained from three centres, two hospitals and a private pathology laboratory, and studied for susceptibility to bacteriophages, resistance to antimicrobial agents and plasmid contents.Results of bacteriophage typing revealed that they belonged to a variety of phage types. Eighteen were untypable by any of the International Set of Phages, 16 belonged to phage group 111, nine to group I, four to group 11, two to group IV and two to the miscellaneous group.The isolates were resistant to one or more of methicillin (Mc), benzyl penicillin (Pc), gentamicin (Gm) , kanamycin (Km) , neomycin (Nm) , streptomycin (Sm) , trimethoprim (Tp), sulphonamides (Su), tetracycline (Tc), minocycline (Mn), chloramphenicol (Cm), novobiocin (Nb) and fusidic acid (Fa). Resistance to Pc was due to the production of beta-lactamase (Bla). No resistance to vancomycin, spectinomycin and erythromycin was detected. Resistance was also found to heavy metals such as cadmium (Cd), mercury (Hg), phenyl mercuric acetate (Pma), arsenate (Asa) and to the nucleic-acid binding compounds propamidine isethionate (Pi) and ethidium bromide (Eb).All but one of the isolates harboured plasmids. The number of plasmids the isolates carried varied from one to six and their sizes ranged from < 1.0 kb to c.48 kb.Location of the resistance determinants was ascertained by curing and transfer experiments. Loss of resistance was tested after growth at 43.5°C and transfer of resistance determinants was attempted by transduction, mixed-culture transfer and conjugation. The results revealed that resistance to Mc, Gm, Tp, Mn and Fa was chromosomal in all the resistant isolates and in some isolates Bla and resistance to Sm and Cd were chromosomal as well as plasmid encoded. In the majority of cases Bla and resistance to Km, Nm, Sm, Tc, Cin, Cd, Hg, Asa, Pma, Pi and Eb was encoded ++ / by plasmids.Conjugation experiments led to the isolation of three unique conjugative plasmids which have not been found to confer resistance to antimicrobials or to produce haemolysins or diffusible pigment (Dip). The three plasmids, pWBG620, pWBG637 and pWBG661, were indistinguishable by restriction endonuclease analysis and DNA-DNA hybridisation. However pWBG620, unlike pWBG637 and pWBG661, was not detected in the cytoplasm of its host and was only detected in transconjugants after it mobilised a non-conjugative Sm-resistance (SmR) plasmid. Further analysis indicated that it is integrated into the chromosome of its host, excises during conjugation and mobilises the SmR plasmid.These plasmids were studied further using pWBG637 as a representative. It was compared with representatives of the two groups of conjugative plasmids which have been reported in the staphylococci. These are the plasmids which encode resistance to Gm, Km and Nm and those which code for the production of diffusible pigment. The three types of conjugative plasmids were compared by restriction endonuclease analysis and DNA- DNA hybridisation and were found to be different. A preliminary restriction map of pWBG637 has also been constructed.However since pWBG637 has no resistance phenotype direct selection for it was not possible in transfer experiments and for incompatibility (Inc.). To study it further it was necessary to construct resistant derivatives which could be selected for in transfer experiments. This was achieved by labelling pWBG637 with resistance transposons to generate two conjugative plasmids, pWBG636 carrying an insert of Tn3851 (Gm- resistance) and pWBG642 carrying an insert of Tn551 (hn- resistance). It was found that transposon labelling had not changed the incompatibility of pWBG637 and therefore pWBG636 and pWBG642 were used in further experiments in place of pWBG637. Inc. ++ / tests with the pWBG637 derivatives revealed that the pWBG637 type of plasmid is not only different from the other two types of conjugative plasmids but is different from any of the described staphylococcal Inc. groups and therefore the pWBG637 type of plasmids represent a new Inc. group 15. The pWBG637 type of plasmids were studied further using plasmids pWBG636 and pWBG642. They were able to transfer conjugatively to a capsulated S.aureus strain either by the polyethylene glycol method or on filter membranes. They also transferred by conjugation to S. epidermidis and Streptococcus faecalis and were able to transfer back from these strains to S.aureus indicating that they also replicate in these hosts. Consequently they have been used to mobilise non-conjugative plasmids from S.epidermidis and non phage typable S.aureus. Both plasmids failed to transfer conjugatively to Bacillus subtilis and Escherichia coli.pWBG637 transferred non-conjugative plasmids by mobilising them in a manner similar to mobilisation (donation) in E.coli or by recombining with them to form new resistance plasmids. In one case, pWBG628 which encodes Bla and resistance to Cd, Km, Nm and Sm and has no homology with pWBG637 recombined with it during conjugation to produce three new conjugative plasmids pWBG629, pWBG630 and pWBG631 carrying resistance determinants from pWBG628. One of these plasmids, pWBG629, was found to be pWBG637 which had acquired a 4.5 kb element encoding resistance to Km, Nm and Sm. This element was shown to be transposable in both rec+ and rec- backgrounds and has been designated Tn3854. It expressed Sm resistance in E.coli and differs on this account from the Gram-negative transposon Tn5 which expresses resistance to Km, Nm and Sm in non-enteric bacteria but only resistance to Km and Nm in E. coli.Where possible the non-conjugative plasmids encoding resistance to ++ / antimicrobial agents were compared with phenotypically similar plasmids isolated from other parts of the world. It was found that the Tc and Sm resistance plasmids were closely related to other plasmids with the same phenotype whereas the Cm resistance plasmids were different.Although the majority of the Bla plasmids belonged to Inc. group 1 they demonstrated significant restriction enzyme fragment length polymorphism when compared with other Bla plasmids.This study has provided the first data on the genetics of antimicrobial resistance in Nigerian S.aureus. Although many of the plasmids studied were found to be similar to those previously described the isolates also contained some unique and previously undescribed plasmids.

This research investigated the mechanism of action in bacteria of 3,4-methylenedioxy-β-nitropropene (BDM-I), a very broad spectrum antimicrobial lead compound in development as an anti-infective drug. The thesis proposes that BDM-I inhibits bacterial protein tyrosine phosphatases, a novel mechanism of action for an antimicrobial agent and a new target in microorganisms. This very open investigation was directed by considerable biological information on the effects of BDM-I in microorganisms and animals which provided insights into possible and improbable cellular targets. The biological effects of BDM-I were investigated using biochemical and cell-based assays, transmission electron microscopy and whole genome DNA microarray analysis. The specific experiments and order of execution were largely dependent on information gained as the project progressed. BDM-I was shown not to target the metabolic pathways of the major classes of antibacterial drugs, which supports a novel mechanism of action. Investigation of several species-specific effects suggested that cell signalling pathways were a possible target. Based on the structure of BDM-I and review of the scientific literature on cell signalling in bacteria, the hypothesis that BDM-I acted by inhibition of protein tyrosine phosphatases (PTP) was supported by demonstrating inhibition of human and bacterial PTP's in an enzyme assay. This mechanism was consistent with other demonstrated effects: inhibition of the intracellular pathogen, Chlamydia trachomatis; inhibition of swarming in Proteus spp. and inhibition of pigment production in Serratia marcescens; and with kill kinetics in bacteria and yeast. A pilot global genome analysis of BDM-I treated Bacillus subtilis did not detect differential expression of PTP genes but has provided many avenues for further investigation. This research further supports the development of BDM-I as a broad spectrum anti-infective drug.

Francisella tularensis is a highly virulent Gram-negative bacterium causing the zoonotic disease tularemia. Arthropod-borne transmission plays an important role in transferring the disease to humans. F. tularensis induces very low amounts of pro-inflammatory cytokines during infection, due to inhibition of immune signaling pathways and an unusual structure of its lipopolysaccharide (LPS). To date, there is no vaccine available that is approved for public use, although an attenuated live vaccine strain (LVS) is commonly used as a model of the more infectious Francisella strains. To produce an effective vaccine it is important to understand the lifecycle of F. tularensis, including the interaction with the arthropod hosts. Drosophila melanogaster is a widely used model organism, which is increasingly being used in host-pathogen interaction studies as the immune pathways in flies are evolutionary conserved to the immune pathways in humans. An important part of the immune defense of D. melanogaster as well as of arthropods in general is the production of antimicrobial peptides. These peptides primarily target the bacterial membrane, inhibiting bacterial proliferation or directly killing the bacteria. The aim of this thesis was to establish D. melanogaster as a model for F. tularensis infection and as a model for arthropod vectors of F. tularensis. Also, to use D. melanogaster to further study the interaction between F. tularensis and arthropod vectors, with specific regard to the host immune signaling and arthropod antimicrobial peptides. F. tularensis LVS infects and kills D. melanogaster in a dose-dependent manner. During an infection, bacteria are found inside fly hemocytes, phagocytic blood cells, similar as in human infections. In mammals genes of the intracellular growth locus (igl) are important for virulence. In this work it is shown that the igl genes are also important for virulence in flies. These results demonstrate that D. melanogaster can be used as a model to study F. tularensis-host interactions. LVS induces a prolonged activation of several immune signaling pathways in the fly, but seem to interfere with the JNK signaling pathway, similarly as in mammals. Overexpression of the JNK pathway in flies has a protective effect on fly survival. Relish mutant flies, essentially lacking a production of antimicrobial peptides, succumb quickly to a F. tularensis infection, however, F. tularensis is relatively resistant to individual D. melanogaster antimicrobial peptides. Overexpressing antimicrobial peptide genes in wildtype flies has a protective effect on F. tularensis infection, suggesting that a combination of several antimicrobial peptides is necessary to control F. tularensis. The production of numerous antimicrobial peptides might be why D. melanogaster survives relatively long after infection. An intact structure of the lipid A and of the Kdo core of Francisella LPS is necessary for resistance to antimicrobial peptides and full virulence in flies. These results are similar to previous studies in mammals. In contrast to studies in mammals, genes affecting the O-antigen of F. tularensis LPS are not necessary for virulence in flies. In conclusion, this thesis work shows that D. melanogaster can be used as a model for studying F. tularensis-host interactions. LVS activates several immune pathways during infection, but interfere with the JNK pathway. Overexpressing the JNK pathway results in increased survival of flies infected with LVS. Despite rather high resistance to individual antimicrobial peptides, exposure to a combination of several D. melanogaster antimicrobial peptides reduces the virulence of F. tularensis.

Understanding how antimicrobial growth promoters (AGPs) affect small intestinal microbiota may help to discover effective alternatives. The impact of dietary supplementation with tylosin phosphate on small intestinal microbial profiles was investigated in growing pigs, and compared with the microbial profile of pigs fed flaxseed or its fractions. Eighteen ileal-cannulated barrows (33.1 +/- 2.4 kg) received either the control diet (C, wheat, peas and soybean meal), or C plus 22 mg/kg tylosin phosphate (T), 20% whole flaxseed (WF), 18% hot-water extracted flaxseed (HWE), 4% flaxseed hulls (H), or 8% flaxseed oil (O) during three 21-d periods in a change over design. Ileal digesta (100 mL) was collected on d 16 and 17 of each period. Two chaperonin-60 universal target (cpn60 UT) libraries were constructed from pooled ileal digesta DNA extracted from the C and T diets. A total of 1634 nucleotide sequences were determined, and 117 different cpn60 UT sequences identified. Microbial diversity was greatest in the C library compared to T. Taxonomic composition between libraries differed, and included Lactobacillales (94% of C and 86% of T sequences), Enterobacteriaceae (3% of C and 13% of T), Clostridiales, Bacillales and Bifidobacterium taxa. T had a reduced ratio of Lactobacillales: Enterobacteriaceae sequences (6:1) compared to C (35:1). Lactobacilli: enterobacteria plate count ratios were highest in WF compared to C or T diets. Lactobacillus johnsonii genomes detected by qPCR were increased by 17.2 and 12%, in T and WF diets, respectively, compared to C. Numbers of L. amylovorus genomes were 25% lower in the H diet compared to C. Numbers of Escherichia coli and Streptococcus alactolyticus genomes were unaffected by dietary treatment, despite differences in library clone frequency for these species. Increased L. johnsonii colonization with tylosin suggests possible probiotic properties of this bacterium. Only inclusion of whole flaxseed resulted in a similar increase in L. johnsonii. Overall, ileal microbial profiles of growing pigs were similar and remained mostly unaffected by dietary tylosin or flaxseed inclusion.

This study describes the pharmacokinetic profile of procaine penicillin G after intraperitoneal (IP) administration in 8 lactating dairy cows. Procaine pencillin G (PPG, 21,000 IU/kg) was deposited into the abdominal cavity of each cow following an incision in the right paralumbar fossa. Blood and milk samples were taken over the following 10 days, at which point the cows were euthanized. Plasma, milk, muscle, liver, and kidney penicillin concentrations were determined by HPLC, with a limit of detection (LOD) of 5 ppb for plasma and milk samples. Noncompartmental methods were used to analyze plasma kinetics. The mean pharmacokinetic parameters (} s.d.) were: Cmax, 5.5 } 2.6 Êg/mL; Tmax, 0.75 } 0.27 h; AUC0-, 10.8 } 4.9 Êg*h/mL; MRT, 2.2 } 0.9 h. All milk from treated cows contained penicillin residues for a minimum of 3 milkings (31 h) and maximum of 5 milkings (52 h) after administration. Concentrations of penicillin G in all muscle, liver, and kidney samples taken 10 days post-administration were below the limit of detection. Necropsy examinations revealed foci of hemorrhage on the rumenal omentum of most cows but peritonitis was not observed. Systemic inflammation as determined by altered leukograms and fibrinogen was noted in one cow. The results of this study demonstrate that IP procaine penicillin G is absorbed and eliminated rapidly in lactating dairy cows.