PEGylating a bacteriophage endolysin inhibits its bactericidal activity.

ABSTRACT: Bacteriophage endolysins (lysins) bind to a cell wall substrate and cleave peptidoglycan, resulting in hypotonic lysis of the phage-infected bacteria. When purified lysins are added externally to Gram-positive bacteria they mediate rapid death by the same mechanism. For this reason, novel therapeutic strategies have been developed using such enzybiotics. However, like other proteins introduced into mammalian organisms, they are quickly cleared from systemic circulation. PEGylation has been used successfully to increase the in vivo half-life of many biological molecules and was therefore applied to Cpl-1, a lysin specific for S. pneumoniae. Cysteine-specific PEGylation with either PEG 10K or 40K was achieved on Cpl-1 mutants, each containing an additional cysteine residue at different locations To the best of our knowledge, this is the first report of the PEGylation of bacteriophage lysin. Compared to the native enzyme, none of the PEGylated conjugates retained significant in vitro anti-pneumococcal lytic activity that would have justified further in vivo studies. Since the anti-microbial activity of the mutant enzymes used in this study was not affected by the introduction of the cysteine residue, our results implied that the presence of the PEG molecule was responsible for the inhibition. As most endolysins exhibit a similar modular structure, we believe that our work emphasizes the inability to improve the in vivo half-life of this class of enzybiotics using a cysteine-specific PEGylation strategy.

Discovery of new intracellular pathogens by amoebal coculture and amoebal enrichment approaches.

Intracellular pathogens such as legionella, mycobacteria and Chlamydia-like organisms are difficult to isolate because they often grow poorly or not at all on selective media that are usually used to cultivate bacteria. For this reason, many of these pathogens were discovered only recently or following important outbreaks. These pathogens are often associated with amoebae, which serve as host-cell and allow the survival and growth of the bacteria. We intend here to provide a demonstration of two techniques that allow isolation and characterization of intracellular pathogens present in clinical or environmental samples: the amoebal coculture and the amoebal enrichment. Amoebal coculture allows recovery of intracellular bacteria by inoculating the investigated sample onto an amoebal lawn that can be infected and lysed by the intracellular bacteria present in the sample. Amoebal enrichment allows recovery of amoebae present in a clinical or environmental sample. This can lead to discovery of new amoebal species but also of new intracellular bacteria growing specifically in these amoebae. Together, these two techniques help to discover new intracellular bacteria able to grow in amoebae. Because of their ability to infect amoebae and resist phagocytosis, these intracellular bacteria might also escape phagocytosis by macrophages and thus, be pathogenic for higher eukaryotes.

Relación de Citoquinas y Biomarcadores como patrones de inflamación y etiología de la neumonía adquirida en la comunidad”

La neumonía adquirida en la comunidad (NAC) es una enfermedad respiratoria infecciosa muy prevalente y de elevada mortalidad que desencadena inflamación sistémica. Las últimas investigaciones analizan la respuesta inflamatoria y han observado que la infección por determinados microorganismos se caracterizaría por un “patrón” de respuesta inflamatoria. Nuestro estudio analizó la diferente etiología de la NAC y la respuesta inflamatoria (PCR, PCT y citocinas). Concluyó que las NAC causadas por microorganismo conocido presentan un “patrón” de inflamación específico (S. pneumoniae eleva la PCT e IL-6, la Legionella aumenta la PCR), y que la respuesta está incrementada en presencia de bacteriemia.

Involvement of Parachlamydia in bovine abortions in Scotland.

Bovine abortion represents a major animal welfare issue and a cause of substantial economic loss yet the rate of successful diagnosis remains low. Chlamydia-related organisms including Parachlamydia have recently emerged as putative cattle abortifacients. Placental tissue samples and fetal lung from bovine abortion submissions across Scotland in Spring 2011 were investigated by histopathology for the presence of suspect Chlamydia-related organisms. Evidence of Chlamydia-related organisms was observed in 21/113 (18.6%) placenta samples. Thirteen of the suspect cases and 18 histopathology negative cases were analysed by molecular and immunohistochemical methods. All samples were PCR positive for Parachlamydia but sequencing revealed high homology between identified environmental 16S sequences in all but three cases. Parachlamydial antigen was detected in 10/31 placental samples (32.2%) with pathology consistent with chlamydial infection. This work supports the need for further surveillance investigations and experimental studies to determine the role of Parachlamydia in bovine abortion.

Cefotaxime acts synergistically with levofloxacin in experimental meningitis due to penicillin-resistant pneumococci and prevents selection of levofloxacin-resistant mutants in vitro.

Cefotaxime, given in two doses (each 100 mg/kg of body weight), produced a good bactericidal activity (-0.47 Deltalog(10) CFU/ml. h) which was comparable to that of levofloxacin (-0.49 Deltalog(10) CFU/ml. h) against a penicillin-resistant pneumococcal strain WB4 in experimental meningitis. Cefotaxime combined with levofloxacin acted synergistically (-1.04 Deltalog(10) CFU/ml. h). Synergy between cefotaxime and levofloxacin was also demonstrated in vitro in time killing assays and with the checkerboard method for two penicillin-resistant strains (WB4 and KR4). Using in vitro cycling experiments, the addition of cefotaxime in sub-MIC concentrations (one-eighth of the MIC) drastically reduced levofloxacin-induced resistance in the same two strains (64-fold increase of the MIC of levofloxacin after 12 cycles versus 2-fold increase of the MIC of levofloxacin combined with cefotaxime). Mutations detected in the genes encoding topoisomerase IV (parC and parE) and gyrase (gyrA and gyrB) confirmed the levofloxacin-induced resistance in both strains. Addition of cefotaxime in low doses was able to suppress levofloxacin-induced resistance.

Estacionalidad de la neumonía adquirida en la comunidad (NAC) y su influencia con el clima

La pneumònia adquirida a la comunitat (PAC) és una patologia molt prevalent, l´etiologia de la qual ve donada per les característiques de la regió geogràfica, de l'agent causal i del pacient. L'estudi de cadascuna d'elles és fonamental per al seu correcte abordatge terapèutic. Ens vam proposar estudiar els canvis de l'agent causal de la PAC en funció de l'estacionalitat i la influència dels canvis climàtics de la nostra àrea geogràfica. Material i mètode: Estudi prospectiu longitudinal de pacients ingressats per PAC des de Gener de 2008 a Desembre de 2009. Analitzem dades sociodemogràfiques, comorbiditat, gravetat, agent etiològic, complicacions i mortalitat. Correlacionem la temperatura mitjana, la precipitació acumulada mitjana i el caràcter de la precipitació per S. pneumoniae i Legionella pneumophila en cada estació de l'any. Anàlisi estadística: Xi quadrat, t de Student per mostres independents, anàlisi de la variància i correlació de Spearman. Resultats: Incloem 155 pacients, 63.9% homes i 54.8% majors de 65 anys. La major incidència de PAC va ser a l'hivern. Streptococcus pneumoniae va ser l'agent causal més freqüent en totes les estacions de l'any a excepció de l'estiu, que va ser Legionella pneumophila. Observem una correlació significativa entre la menor temperatura mitjana estacional i l'etiologia pneumocòccica i al revés quan l'agent causal va ser Legionella pneumophila. No obstant això, no trobem diferències etiològiques per estacions en relació amb la humitat ambiental. Conclusions: En la nostra àrea, Streptococcus pneumoniae és l'agent etiològic més freqüent a l'hivern amb baixes temperatures mentre que a l'estiu, amb altes temperatures, és Legionella pneumophila.

HIV and STI surveillance in Northern Ireland 2010: An analysis of data for the calendar year 2009

This annual analysis of data provides an overview of HIV and STI epidemiology in Northern Ireland for the calendar year 2009. Information from a variety of sources is collated and analysed in detail, while any evident trends over time are highlightedwithgraphs and tables. As well as a general summary of STI diagnoses and a number of overall conclusions, the report looks specifically at each of the following STIs: chlamydia, gonorrhoea, genital herpes, genital warts, syphilis, lymphogranuloma venereum (LGV) and HIV.

Ceftriaxone acts synergistically with levofloxacin in experimental meningitis and reduces levofloxacin-induced resistance in penicillin-resistant pneumococci.

Ceftriaxone acted synergistically with levofloxacin in time-killing assays in vitro over 8 h against two penicillin-resistant pneumococcal strains (WB4 and KR4; MIC of penicillin: 4 mg/L). Synergy was confirmed with the chequerboard method, showing FIC indices of 0.25. In the experimental rabbit meningitis model, ceftriaxone (1x 125 mg/kg) was slightly less bactericidal (-0.30 Deltalog(10) cfu/mL(.)h) compared with levofloxacin (-0.45 Deltalog(10) cfu/mL(.)h) against the penicillin-resistant strain WB4. The combination therapy (levofloxacin and ceftriaxone) was significantly superior (-0.64 Deltalog(10) cfu/mL(.)h) to either monotherapy. In cycling experiments in vitro, the addition of ceftriaxone at a sub-MIC concentration (1/16 MIC) reduced levofloxacin-induced resistance in the two strains KR4 and WB4. After 12 cycles with levofloxacin monotherapy, the MIC increased 64-fold in both strains versus a 16-fold increase with the combination (levofloxacin + ceftriaxone 1/16 MIC). In both strains, levofloxacin-induced resistance was confirmed by mutations detected in the genes parC and gyrA, encoding for subunits of topoisomerase IV and gyrase, respectively. The addition of ceftriaxone suppressed mutations in parC but led to a new mutation in parE in both strains.

Antimicrobial activity and chemical investigation of Brazilian Drosera

The antimicrobial activity of three different extracts (hexanic, ethyl acetate, methanol) obtained from Brazilian Drosera species (D. communis, D. montana var. montana, D. brevifolia, D. villosa var. graomogolensis, D. villosa var. villosa, Drosera sp. 1, and Drosera sp. 2 ) were tested against Staphylococcus aureus (ATCC 25923), Enterococcus faecium (ATCC23212), Pseudomonas aeruginosa (ATCC27853), Escherichia coli (ATCC11229), Salmonella choleraesuis (ATCC10708), Klebsiella pneumoniae (ATCC13883), and Candida albicans (a human isolate). Better antimicrobial activity was observed with D. communis and D. montana var. montana ethyl acetate extracts. Phytochemical analyses from D. communis, D. montana var. montana and D. brevifolia yielded 5-hydroxy-2-methyl-1,4-naphthoquinone (plumbagin); long chain aliphatic hydrocarbons were isolated from D. communis and from D. villosa var. villosa, a mixture of long chain aliphatic alcohols and carboxylic acids, was isolated from D. communis and 3b-O-acetylaleuritolic acid from D. villosa var. villosa.

HIV and STI surveillance in Northern Ireland 2011: An analysis of data for the calendar year 2010

This annual analysis of data provides an overview of HIV and STI epidemiology in Northern Ireland for the calendar year 2010. Information from a variety of sources is collated and analysed in detail, while any evident trends over time are highlighted�with�graphs and tables. As well as a general summary of STI diagnoses and a number of overall conclusions, the report looks specifically at each of the following STIs: chlamydia, gonorrhoea, genital herpes, genital warts, syphilis, lymphogranuloma venereum (LGV) and HIV.

Been given any good gifts recently? (Female)

This poster draws attention to the health risks of chlamydia for women and advises on the use of condoms as a method of protection from sexually transmitted infections (STIs). It also provides contact details for the�Genito Urinary Medicine (GUM) clinics in Northern Ireland.

Been given any good gifts recently? (Male)

This poster draws attention to the health risks of chlamydia for men and advises on the use of condoms as a method of protection from sexually transmitted infections (STIs). It also provides contact details for the Genito Urinary Medicine (GUM) clinics in Northern Ireland.

One of the most popular brands of sexually transmitted infection

This poster informs about chlamydia, stating that it doesn't always show symptoms and can be prevented by using a condom. It also provides updated attendance and contact details for the�Genito Urinary Medicine (GUM) clinics in Northern Ireland.

One of the most popular brands of sexually transmitted infection

This poster informs about chlamydia, stating that it doesn't always show symptoms and can be prevented by using a condom. It also provides updated attendance and contact details for the�Genito Urinary Medicine (GUM) clinics in Northern Ireland.

HIV and STI surveillance in Northern Ireland 2010: An analysis of data for the calendar year 2009

This annual analysis of data provides an overview of HIV and STI epidemiology in Northern Ireland for the calendar year 2009. Information from a variety of sources is collated and analysed in detail, while any evident trends over time are highlighted�with�graphs and tables. As well as a general summary of STI diagnoses and a number of overall conclusions, the report looks specifically at each of the following STIs: chlamydia, gonorrhoea, genital herpes, genital warts, syphilis, lymphogranuloma venereum (LGV) and HIV.