Staphylococcus aureus d'origine communautaire (SARM-C) est responsable d'infections sévères comme la pneumonie nécrosante. La leucocidine de Panton-Valentine (PVL), l'Hémolysine-α(Hla) et la protéine A (Spa) sont les facteurs de virulence impliqués dans cette infection. L'analyse bibliographique montre que les polynucléaires neutrophiles (PNN) sont la principale cible de la PVL et que les α-défensines secrétées par les PNN peuvent neutraliser des toxines bactériennes. De plus, il est montré que des antibiotiques modulent la sécrétion des facteurs de virulence par S. aureus. Dans ce contexte, nous avons étudié l'effet des défensines sur la cytotoxicité de la PVL ainsi que l'effet des antibiotiques seuls et couplés aux défensines sur la production de PVL, Hla et Spa par des SARM-C. Nous montrons pour la première fois que les défensines peuvent diminuer la cytotoxicité de la PVL. Dans la deuxième partie du travail personnel, nous avons observé que l'effet de faibles concentrations d'antibiotique sur les SARM- C dépendait de l'antibiotique et du facteur de virulence. La clindamycine et le linézolide diminuent la PVL, Hla et Spa alors que la tigécycline supprime essentiellement la production de PVL. La daptomycine et la vancomycine n'ont pas d'effet. Nous avons démontré que l'effet des α-défensines couplé aux antibiotiques sur la virulence concerne essentiellement Spa. De façon intéressante, les α-défensines couplé aux antibiotiques ne diminuent pas la production de Spa pour le clone USA300 contrairement aux autres SARM-C, ce qui pourrait expliquer la sévérité de ses infections et son succès épidémique / Community Acquired Methicillin Resistant Staphylococcus aureus (CA-MRSA) induce severe diseases such as necrotizing pneumonia. Panton-Valentine Leucocidin (PVL), Hémolysine-α(Hla) and protein A (Spa) are important virulence factors involved in this infection. The literature review shows that polymorphonuclear neutrophils (PMNs) are the target cells of PVL action in lungs and α-defensins secreted by PMNs have the capability to neutralize bacterial toxins. Moreover, low concentrations of antibiotic can modulate virulence factor production by S. aureus. In this context, the goal of this work was to study the impact of defensins on various cytotoxicity effects induced by PVL on human PMNs and to study the effect of antibiotics combined with defensins on the production of PVL, Hla and Spa by CA-MRSA. We show for the first time that α-defensins can decrease PVL toxicity mainly interacting with pore formation. Secondly, we show that low concentrations of antibiotic s’ effect on CA-MRSA virulence expression depend on the antibiotic and the virulence factor. Clindamycin and linezolid consistently suppress the expression of PVL, Hla and Spa by CA-MRSA, whereas tigecycline specifically suppress PVL expression. Daptomycin and vancomycin have no significant effects at these concentrations. For the first time, we demonstrate that the modulatory effect of α-defensins on virulence, in conjunction with antibiotics, mainly regards Spa production. Interestingly, antibiotics and defensins do not decrease Spa expression in USA300, unlike in other CA-MRSA clones. It could explain the severity of USA300 related infections and the spreading success of this CA-MRSA lineage

The recent rise of multidrug resistant microorganisms has grown from an isolated concern to a massive public health crisis. It has become imperative that scientists look for new ways to combat this issue. Due to the selective pressures of competition, bacteria and other microbes possess a host of defenses and weapons designed to exploit vulnerabilities in other microorganisms. Consequently, the study of these systems and microbial interactions has much to reveal in the search for novel antimicrobial treatments. Previous research from our laboratory has discovered that both Alcaligenes faecalis and Alcaligenes viscolactis, two rarely studied and generally non-virulent bacteria, exert a microbicidal effect on Candida albicans and Staphylococcus aureus, two pathogenic and frequently drug-resistant organisms. In this study, we confirmed that these effects are via a live-cell, contact-dependent mechanism and showed that both Alcaligenes species inhibit S. aureus at the attachment phase of biofilm growth. Additionally, we found that A. faecalis and A. viscolactis target Gram-positive bacteria outside the genus Staphylococcus and certain Gram-negative species as well as Candida glabrata. This study also provides novel evidence of a putative Type VI Secretion System in both Alcaligenes species, which may explain their antimicrobial phenotype. Despite efforts to identify the genetic elements involved via mutagenesis, the mechanism of these interactions remain elusive due to the difficulty of gene transfer in these organisms. We hope these results will increase current knowledge of Alcaligenes’ capabilities and genetic composition as well as establish the groundwork for future efforts to discover its inhibitory system and mechanisms.

Chlamydia trachomatis, the most common bacterial sexually transmitted disease agent worldwide, enters a viable, non-dividing and non-infectious state (historically termed persistence and more recently referred to as the chlamydial stress response) when exposed to penicillin G in culture. Notably, penicillin G-exposed chlamydiae can reenter the normal developmental cycle upon drug removal and are resistant to azithromycin-mediated killing. Because penicillin G is less frequently prescribed than other ß-lactams, the clinical relevance of penicillin G-induced chlamydial persistence/stress has been questioned. The goal of this study was to determine whether more commonly used penicillins also induce C. trachomatis serovar E persistence/stress. All penicillins tested, as well as clavulanic acid, induced formation of aberrant, enlarged reticulate bodies (RB) (called aberrant bodies or AB) characteristic of persistent/stressed chlamydiae. Exposure to the penicillins and clavulanic acid also reduced chlamydial infectivity by >95%. None of the drugs tested significantly reduced chlamydial unprocessed 16S rRNA or genomic DNA accumulation, indicating that the organisms were viable, though non-infectious. Finally, recovery assays demonstrated that chlamydiae rendered essentially non-infectious by exposure to ampicillin, amoxicillin, carbenicillin, piperacillin, penicillin V, and clavulanic acid recovered infectivity after antibiotic removal. These data definitively demonstrate that several commonly used penicillins induce C. trachomatis persistence/stress at clinically relevant concentrations.

Staphylococcus aureus has developed resistance to several antibiotics including vancomycin, which is often used as a “last resort” treatment. There is an ever-increasing need to develop novel antimicrobial treatments to combat S. aureus and other drug resistant bacteria. Microorganisms are most often found in polymicrobial communities where they either exhibit synergistic or antagonistic relationships. Competition between microorganisms can lead to the discovery of new antimicrobial targets as the specific mechanisms of resistance are elucidated. In addition, synergistic treatments are being evaluated for their combined effect and potential to decrease the concentration of drugs needed, and thus the side effects also. Alcaligenes faecalis is a microorganism that our lab has previously shown to inhibit S. aureus and other various bacterial species. In this study, we found that A. faecalis reduces the planktonic growth of S. aureus by 94.5% and biofilm growth by 76.6%. A. faecalis also has a synergistic effect when paired with bacitracin to reduce the planktonic growth by 99.9% and biofilm growth by 99.7%. Transposon mutagenesis was successfully performed on A. faecalis, and loss of function mutations were attained. Two mutants were no longer able to inhibit the growth of Staphylococcus aureus, Candida albicans, or Bacillus megaterium. Further analysis and genomic sequencing of these mutants is needed to determine the gene(s) that were interrupted and the mechanism of A. faecalis’ antimicrobial activity. The findings of this study may aid in the identification of new therapeutic targets for novel S. aureus treatments.

Amicoumacin A (Ami) halts bacterial growth by inhibiting the ribosome during translation. The Ami binding site locates in the vicinity of the E-site codon of mRNA. However, Ami does not clash with mRNA, rather stabilizes it, which is relatively unusual and implies a unique way of translation inhibition. In this work, we performed a kinetic and thermodynamic investigation of Ami influence on the main steps of polypeptide synthesis. We show that Ami reduces the rate of the functional canonical 70S initiation complex (IC) formation by 30-fold. Additionally, our results indicate that Ami promotes the formation of erroneous 30S ICs; however, IF3 prevents them from progressing towards translation initiation. During early elongation steps, Ami does not compromise EF-Tu-dependent A-site binding or peptide bond formation. On the other hand, Ami reduces the rate of peptidyl-tRNA movement from the A to the P site and significantly decreases the amount of the ribosomes capable of polypeptide synthesis. Our data indicate that Ami progressively decreases the activity of translating ribosomes that may appear to be the main inhibitory mechanism of Ami. Indeed, the use of EF-G mutants that confer resistance to Ami (G542V, G581A, or ins544V) leads to a complete restoration of the ribosome functionality. It is possible that the changes in translocation induced by EF-G mutants compensate for the activity loss caused by Ami. / Russian Foundation for Basic Research / Revisión por pares

The purpose of this study is to find out that if antibiotic streptomycin decreases neuronal network activity or affects the pharmacological responses. The experiments in this study were conducted via MEA (multi-electrode array) technology which records neuronal activity from devices that have multiple small electrodes, serve as neural interfaces connecting neurons to electronic circuitry. The result of this study shows that streptomycin lowered the spike production of neuronal network, and also, sensitization was seen when neuronal network pre-exposed to streptomycin.

V průběhu projektu byly vyvinuty unikátní kolagenní pěny z kolagenu získaného z kůže sladkovodní ryby (kapr obecný, Cyprinus carpio). Pomocí síťování karbodiimidem byl překonán problém s nestabilitou kolagenní matrix z kolagenu získávaného z chladnokrevných živočichů při tělesné teplotě savců. Následně byly pěny impregnovány antibiotiky (gentamicin a vankomycin) a opětovně lyofilizovány, což je postup, který zajišťuje požadovanou koncentraci antibiotika bez rizika následného vymytí při dalších technologických krocích. Uvedený produkt je, na rozdíl od přípravků z nesíťovanéhokolagenu, stabilní i při sterilizaci gamma zářením. Finální sterilizovaný produkt byl testován in vivo na potkaním modelu infikované rány. Byla prokázána efektivita v léčbě potenciálně letální infekce Pseudomonas aeruginosa a kmene Stafylococcus aureus rezistentní k meticilinu (MRSA). Vzhledem k vysoké potřebě profylaxe a terapie infekcí pooperačních a jiných ran právě výše uvedenými polyrezistentními původci se jedná o slibný prostředek k budoucímu klinickému využití. Zkušenosti, které jsme získali v průběhu uvolnování ATB z kolagenních pěn budou v dalším vyvoji použity pro impregnaci zevní kolagenní vrstvy cévní protézy, čímž bychom mohli eliminovat jednu z největších nevýhod a rizik spojených s použitím umělých materiálu a tím je...

Antibiotic Feed Additives (AFA) have been used to prevent many bacterial infections during weaning of livestock. The use of these AFA’s resulted in the development of multiresistant bacterial strains and was therefore banned by the European Union. The United States also restricted the use of these feed additives considerably. Many scientists have started to search for alternatives in the prophylactic and therapeutic treatment of bacterial infections. Plants have been used traditionally by resource poor people all over the world to treat many infections. Diarrhoea not only causes many problems in the high intensity production of livestock but also leads to many human deaths. A large number of plants have been used to treat diarrhoea in humans and animals. Several authors have selected one or two species based on traditional use to evaluate in depth. In this project several different relevant parameters of 53 plant species used traditionally to treat diarrhoea were investigated in order to develop a model that would identify the species with the highest chance of delivering a useful antibacterial product. Antibacterial activities against two pathogens important in diarrhoea (Staphylococcus aureus and Escherichia coli) were positive parameters in selecting species. Because tannins frequently have antibacterial activity, but are not useful as prophylactic agents due to their effect on production, high tannin content was considered to be a negative indication. Cellular toxicity was also used as a negative parameter at a later stage. In addition to the in vitro assays there are also other parameters to be investigated to evaluate the potential use of plants. The influence of season of collection was determined on the antibacterial activity and tannin content of acetone leaf extracts of five plant species traditionally used to treat diarrhoea. They were Acacia karroo, Acacia sieberiana var. woodii, Peltophorum africanum, Trichilia emetica and Ziziphus mucronata. The antibacterial activity varied depending on the season of collection with the best activity generally in the months of late summer to autumn (January to April). The activity of Acacia karoo against E. coli was best in the month of April (MIC = 0.11 mg/ml (TA = 332 ml/g)) and against S. aureus in the month of March (MIC = 0.06 mg/ml (TA = 334 ml/g)). Acacia sieberiana subsp. woodii extracts had the best activity against E. coli (MIC = 0.10 mg/ml (TA = 303 ml/g)) in March against S. aureus in April (MIC = 0.08 mg/ml (TA = 303 ml/g)). <i.Peltophorum africanum extracts were most active against E. coli in February (MIC = 0.05 mg/ml (TA = 1188 ml/g)) and against S. aureus in February and March (MIC = 0.04 mg/ml (TA = 1188 ml/g and 1075 ml/g)). Trichilia emetica extracts were generally not very active against the bacterial strains (best activity: MIC = 0.22 mg/ml (TA = 74 ml/g) against E. coli in May and MIC = 0.28 mg/ml (TA = 26 ml/g) against S. aureus in December). Ziziphus mucronata was most active against both bacterial strains and in May (E. coli: MIC = 0.10 mg/ml (TA = 589 ml/g); S. aureus: MIC = 0.04 mg/ml (TA = 1099 ml/g)). The tannin content varied in the extracts as well. The antibacterial activity however did not seem to be directly correlated to the tannin content. Another important parameter in the use of plant species is to determine the interspecies variation of plants based on genetic or environmental influences. Leaves from 42 plants of Combretum molle were collected at different locations during the same season. The average MIC against E. coli was 0.227 mg/ml. The low standard deviation of 0.07 indicates that there was very little variation in activity. The average value against S. aureus was 0.399 mg/ml with a slightly higher standard deviation of 0.16. However due to the fact that the samples from different areas extracted different amounts, the total activity varied. The tannin assays revealed that there was with one exception no correlation between the antibacterial activity and the tannin content. So it can be safe to assume that genetic variation does not influence the activity too much at least in C. molle leaves Fifty three plant species traditionally used to treat diarrhoea in published literature were then ranked using a novel system in order to determine which species had the most potential value. Ranking was based on the lowest MIC value against E. coli, lower activity against S. aureus (to limit selecting for general metabolic toxins), low tannin concentration and high extract yield. From this ranking, five plants were chosen to investigate their potential value further: Acacia sieberiana var. woodii (E. coli: MIC = 0.13 mg/ml, TA = 108 ml/g; S. aureus: MIC = 0.13 mg/ml, TA = 108 ml/g; Yield = 14 mg), Albizia adianthifolia (E. coli: MIC = 0.14 mg/ml, TA = 239 ml/g; S. aureus: MIC = 0.04 mg/ml, TA = 765 ml/g; Yield = 34 mg), Deinbollia oblongifolia (E. coli: MIC = 0.17 mg/ml, TA = 158 ml/g; S. aureus: MIC = 0.08 mg/ml, TA = 338 ml/g; Yield = 27 mg), Spirostachys africana (E. coli : MIC = 0.13 mg/ml, TA = 300 ml/g; S. aureus: MIC = 0.09 mg/ml, TA = 438 ml/g; Yield = 38 mg) and Tetradenia riparia (E. coli : MIC = 0.09 mg/ml, TA = 214 ml/g; S. aureus: MIC = 0.13 mg/ml, TA = 149 ml/g; Yield = 20 mg). None of the plants contained any tannin. The next step towards the recommendation of a plant for the development of a commercial product was to evaluate the cytotoxicity of the selected five species. The following values were obtained: Acacia sieberiana var. woodii LC50 = 0.026 mg/ml, Albizia adianthifolia LC50 = 0.068 mg/ml, Deinbollia oblongifolia LC50 = 0.078 mg/ml, Spirostachys africana LC50 = 0.025 mg/ml and Tetradenia riparia LC50 = 0.028 mg/ml. Deinbollia oblongifolia (for its low LC50 value) and Spirostachys africana (for its good antibacterial activity and total activity) were potentized by removing inactive compounds through solvent-solvent fractionation. The antibacterial activity against E. coli was increased this way (MIC = 0.08 mg/ml for Deinbollia oblongifolia (chloroform fraction) and MIC = 0.08 mg/ml for Spirostachys africana (chloroform fraction)) The LC50 values for both chloroform fractions were determined (LC50 = 0.188 mg/ml for Deinbollia oblongifolia and LC50 = 0.062 mg/ml for Spirostachys africana ). The selectivity index (SI) was also determined and proved that the potentization was indeed successful (Deinbollia oblongifolia SI = 2.35 compared to a value of 0.45 for the crude extract; Spirostachys africana SI = 0.78 compared to a value of 0.19 for the crude extract). Based on these values, the chloroform fraction of Deinbollia oblongifolia was chosen as the less toxic one with similar activity and a higher selectivity index to be worked on further. One of the active compounds was isolated and evaluated for its activity against E. coli (MIC = 0.74 mg/ml) The LC50 value of 0.042 mg/ml indicated that the activity of the extract was a result of synergism rather than being due to a single active compound (the selectivity index (SI) was 0.06 compared to the values of 0.45 for the crude and 2.35 for the potentized extract). The plant extracts should of course be as effective against pathogenic strains as they were against the ATCC strains and so the extracts and pure compound of Deinbollia oblongifolia were tested for their activity against four different pathological E. coli strains. The results showed that the crude extract and the fraction were as active as in the preliminary screening results against only one of the four pathological strains. The pure compound on the other hand was more active against all four pathological strains than against the ATCC strain. The next step was to test the safety of the extracts of Deinbollia oblongifolia in mammals. Unfortunately neither the crude extract nor the chloroform fraction of Deinbollia oblongifolia could be used safely in a living organism or in an isolated organ study. A part of the problem may have been caused by the vehicle used in the study despite reports in the literature that an acetone water mixture is safe to use. In general all the species investigated had good antibacterial activity against E. coli this supports the traditional use of these species although we used acetone as extractant rather than the water used traditionally. There were major differences in antibacterial activity over a season indicating that mature leaves were more active than young leaves before senescence started. At least in the case of Combretum molle there was little difference in the antibacterial activity of many plants collected at different locations during the same season. The results obtained in this study could be useful in further studies to develop extracts that can be used to control diarrhoea in animals. Possibly more emphasis should be put on the difference in activity towards E. coli and S. aureus to eliminate the presence of general metabolic toxins. Such an approach would lead to a different priority order for species to examine. A major first step would probably be to test the in vitro and in vivo toxicity of selected species. / Thesis (PhD)--University of Pretoria, 2010. / Paraclinical Sciences / unrestricted

Pathogens such as <i>Listeria</i>, <i>Shigella</i>, <i>Brucella</i>, <i>Salmonella</i>, <i>Mycobacterium tuberculosis</i> and <i>methicillin-resistant Staphylococcus aureus</i> (MRSA) can traverse into mammalian cells, such as phagocytic macrophages. Once inside, these bacteria can survive and reproduce, causing chronic infections. It is of utmost importance to develop novel antibiotics with broad spectrum activity to control these deadly bacteria. Broad spectrum activity will allow for targeting of pathogens with different structures and cell membrane components.<div>This work focuses on the synthesis of a dual antibiotic agent, composed of a cationic amphiphilic polyproline helix (CAPH) possessing cell penetrating and nonmembrane lytic antimicrobial capabilities (P14LRR), and a derivative of the polymyxin B (PMX) antibacterial peptide. This dual antibiotic conjugate was created to be a tool to potentially clear intracellular pathogenic bacteria. Overall, the reduction of the disulfide bond linking the two antibiotics within the reducing environment of cells would release the individual antimicrobial agents, and could have improved cell membrane penetration and intracellular synergistic activity. Herein, the synthesis of the dual antibiotic agent, P14LRR-PMX, is discussed. </div>

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