Characterization of a Peptidoglycan-Degrading Protein: Biochemical and Antimicrobial Characteristics, Antibiotic Synergism, and Delivery System Innovation.

The global health threat posed by antibiotic resistance has led to new research involving bacteriophage-encoded enzymes. This study characterized a new peptidoglycan-degrading protein and evaluated its synergism with colistin and its antimicrobial efficacy when conjugated with polycationic-polymer nanoparticles. The gene that codes for endolysin in the vB_PaeM_USP2, a Pseudomonas aeruginosa bacteriophage, was cloned and expressed in Escherichia coli. The recombinant endolysin (rEnd2) was purified and its biochemical properties were determined using peptidoglycan substrate. The enzymatic activity was measured through peptidoglycan layer degradation and a decrease in turbidity of permeabilized Gram-negative bacteria. The antimicrobial activity of rEnd2, alone and in combination with colistin, was evaluated by checkerboard assay. The antibacterial activity of the cationic lipid oleylamine (OAM) conjugated with rEnd2 (OAM-rEnd2) was evaluated by time killing assay. The rEnd2 is structurally analogue with other endolysins and showed muramidase activity. The rEnd2 maintained higher activity between pH 6.0 to 7.5, had maximum activity at 35 °C, and was not affected by chaotropic and reducing reagents. It was sensitive to an increase in surfactant concentration, being inactivated by sodium dodecyl sulfate and cetyltrimethylammonium bromide. Ions exhibited neither a positive nor a negative effect on enzyme activity. The rEnd2 showed clear muralytic activity and decreased turbidity of permeabilized Gram-negative bacteria. However, it did not control bacterial growth despite the combination with an antibiotic and its complexation with polycation (OAM-rEnd2 nanoparticle conjugate). The rEnd2 did not show clear antimicrobial activity suggesting further optimization of conditions for its activity or engineering and modification.

Effect of effervescent tablets on removable partial denture hygiene.

PURPOSE: To evaluate the effectiveness of five alkaline peroxide-based effervescent tablets in reducing both biofilms and the food layer adhered on the cobalt-chromium surface. METHODS: Cobalt-chromium metal alloy specimens were contaminated with Candida albicans, Candida glabrata, Streptococcus mutans and Staphylococcus aureus. After biofilm maturation, the specimens were immersed in Polident 3 Minute, Polident for Partials, Efferdent, Steradent, Corega Tabs or distilled water (control). Residual biofilm rates were determined by colony forming units counts and biofilm biomass. In parallel, to investigate the denture cleaning capability of effervescent tablets, artificially contaminated removable partial dentures were treated with each cleanser. Data were analyzed by Kruskal-Wallis followed by Dunn post hoc test or ANOVA followed by Tukey post hoc test (α= 0.05). RESULTS: None of the hygiene solutions reduced C. albicans biofilm. Efferdent and Corega Tabs promoted reduction of C. glabrata biofilm, while Steradent was favorable against S. aureus biofilm. For S. mutans, lower biofilm rates were observed after immersion in Polident for Partials and Steradent. The effervescent tablets showed good cleaning performance, removing an artificial layer with carbohydrates, proteins, and fats, however, they were not effective in removing aggregated mature biofilm. CLINICAL SIGNIFICANCE: The different effervescent tablets presented favorable antimicrobial activity against C. glabrata, S. mutans and S. aureus on cobalt-chromium surfaces and showed cleaning capability. However, for an appropriate biofilm control, a complementary method should be evaluated since none of the peroxide-based solutions reduced C. albicans biofilms or substantially removed aggregated biofilm.

Identification and Characterization of New Bacteriophages to Control Multidrug-Resistant Pseudomonas aeruginosa Biofilm on Endotracheal Tubes.

Studies involving antimicrobial-coated endotracheal tubes are scarce, and new approaches to control multidrug-resistant Pseudomonas aeruginosa biofilm on these devices should be investigated. In this study, five new P. aeruginosa bacteriophages from domestic sewage were isolated. All of them belong to the order Caudovirales, Myoviridae family. They are pH and heat stable and produce 27 to 46 particles after a latent period of 30 min at 37°C. Their dsDNA genome (ranging from ∼62 to ∼65 kb) encodes 65 to 89 different putative proteins. They exhibit a broad lytic spectrum and infect 69.7% of the P. aeruginosa strains tested. All the bacteriophages were able to reduce the growth of P. aeruginosa strains in planktonic form. The bacteriophages were also able to reduce the biofilm viability rates and the metabolic activity of P. aeruginosa strains in a model of biofilms associated with endotracheal tubes. In addition, scanning electron microscopy micrographs showed disrupted biofilms and cell debris after treatment of bacteriophages, revealing remarkable biofilm reduction. The lytic activity on multidrug-resistant P. aeruginosa biofilm indicates that the isolated bacteriophages might be considered as good candidates for therapeutic studies and for the application of bacteriophage-encoded products.

Frequency and carbapenems susceptibility profile of non-fermenting Gram-negative bacilli isolated from clinical samples between 2007 and 2012 / Frequência e perfil de suscetibilidade aos carbapenêmicos de bastonetes Gram-negativos não fermentadores de glicose isolados de amostras clínicas entre 2007 e 2012

ABSTRACT Introduction: One of the major problems in health services is the occurrence of healthcare-associated infections (HAIs) by microorganisms resistant to various antimicrobials. Objectives: To describe the frequency and susceptibility profile of Pseudomonas aeruginosa and Acinetobacter baumannii to carbapenems in the hospital from Fundação Santa Casa de Franca, São Paulo, Brazil. Methods: The susceptibility of P. aeruginosa and A. baumannii to carbapenems from 304 clinical isolates between 2007 and 2012 was retrospectively analyzed from a microbiology database at the clinical laboratory of the hospital of Fundação Santa Casa de Franca, São Paulo, Brazil. Results: From isolated and identified strains, 236 (5.3%) P. aeruginosa were susceptible to imipenem (2007 - 69.6% to 2012 - 41.7%) and meropenem (2007 - 63.3% to 2012 - 25%). In addition, all 68 (1.7%) A. baumannii isolates were susceptible to both antibiotics. Conclusion: A. baumannii resistance to carbapenems was not identified; however, there was a decrease in susceptibility to carbapenems over the years for P. aeruginosa.

RESUMO Introdução: Um dos grandes problemas nos serviços de saúde é a ocorrência de infecções relacionadas com assistência à saúde (IRAS) por microrganismos resistentes a vários antimicrobianos. Objetivos: Descrever a frequência e o perfil de suscetibilidade de Pseudomonas aeruginosa e Acinetobacter baumannii aos carbapenêmicos no hospital da Fundação Santa Casa de Franca, São Paulo, Brasil. Métodos: Retrospectivamente, a suscetibilidade de P. aeruginosa e A. baumannii aos carbapenêmicos foi analisada em 304 isolados clínicos entre 2007 e 2012, a partir de um banco de dados do setor de microbiologia do laboratório clínico do hospital da Fundação Santa Casa de Franca, São Paulo, Brasil. Resultados: Das cepas isoladas e identificadas, 236 (5,3%) P. aeruginosa eram suscetíveis a imipenem (2007 - 69,6% a 2012 - 41,7%) e meropenem (2007 - 63,3% a 2012 - 25%). Além disso, todos os 68 (1,7%) isolados de A. baumannii eram suscetíveis aos dois antibióticos. Conclusão: Não foi identificada resistência de A. baumannii aos carbapenêmicos, no entanto houve diminuição da suscetibilidade aos carbapenêmicos no decorrer dos anos para P. aeruginosa.

Evaluation of a protocol for reducing the microbial contamination of dental unit water.

Biofilm on dental unit waterlines can spread microbial contamination in the water. The aim of this study was to investigate microbial contamination of water from supplies and dental units before and after the implementation of a protocol for microbial quality improvement and maintenance of dental unit water. The microbial load was evaluated in water from 27 taps and dental units (reservoirs, air-water syringes and highspeed outputs without handpieces) using the PetrifilmT system (total aerobic bacteria and fungi) and conventional culture media (enterobacteria and Legionella spp.). The bacterial load in water samples from taps and reservoirs was within the parameter established by Brazilian legislation (<500CFU/mL); but the bacterial load in samples from air-water syringes and high-speed outputs without handpieces was not. The implementation of the protocol for the maintenance of microbial quality in dental unit water reduced bacterial load in highspeed outputs without handpieces (p=0.004). Enterobacteria and Legionella spp. were not isolated from any of the water samples from taps and dental units.

Biofilme nas linhas d\'água de equipos odontológicos pode propagar contaminando microbiana na água. O objetivo deste estudo foi investigar a contaminando microbiana da água de abastecimentos e equipos odontológicos antes e após a implementando de um protocolo para melhoria e manutenndo da qualidade microbiològica da água de equipos dontológicos. Avaliou-se a carga microbiana da água de 27 torneiras e equipos (reservatórios, seringas tríplice e alta rotando sem as penas de mdo) de uma clínica odontológica por meio do sistema PetrifilmT (bacterias aeróbias totais e fungos) e meios de cultura convencionais (enterobactérias e Legionella spp.). A carga bacteriana em amostras de água das torneiras e reservatórios estava dentro do paràmetro estabelecido pela legislando brasileira (<500 UFC/mL), mas a carga bacteriana das seringas tríplices e das saídas dos alta rotando sem as penas de mdo ndo estava. A implementando do protocolo para manutenndo da qualidade da água dos equipos reduziu a carga bacteriana nas saídas dos alta rotando sem as penas de mdo (p=0,004). Enterobactérias e Legionella spp. ndo foram isoladas de qualquer das amostras de água das torneiras e dos equipos odontológicos.

Evaluation of a protocol for reducing the microbial contamination of dental unit water

Biofilm on dental unit waterlines can spread microbial contamination in the water. The aim of this study was to investigate microbial contamination of water from supplies and dental units before and after the implementation of a protocol for microbial quality improvement and maintenance of dental unit water. The microbial load was evaluated in water from 27 taps and dental units (reservoirs, airwater syringes and highspeed outputs without handpieces) using the Petrifilm™ system (total aerobic bacteria and fungi) and conventional culture media (enterobacteria and Legionella spp.). The bacterial load in water samples from taps and reservoirs was within the parameter established by Brazilian legislation (<500CFU/mL); but the bacterial load in samples from airwater syringes and highspeed outputs without handpieces was not. The imple mentation of the protocol for the maintenance of microbial quality in dental unit water reduced bacterial load in highspeed outputs without handpieces (p=0.004). Enterobacteria and Legionella spp. were not isolated from any of the water samples from taps and dental units (AU)

Biofilme nas linhas d'água de equipos odontológicos pode propagar contaminação microbiana na água. O objetivo deste estudo foi investigar a contaminação microbiana da água de abastecimentos e equipos odontológicos antes e após a implemen tação de um protocolo para melhoria e manutenção da qualidade microbiológica da água de equipos odontológicos. Avaliouse a carga microbiana da água de 27 torneiras e equipos (reservatórios, seringas tríplice e alta rotação sem as peças de mão) de uma clínica odontológica por meio do sistema Petrifilm™ (bactérias aeróbias totais e fungos) e meios de cultura convencionais (enterobactérias e Legionella spp.). A carga bacteriana em amostras de água das torneiras e reservatórios estava dentro do parâmetro estabelecido pela legislação brasileira (<500 UFC/mL), mas a carga bacte riana das seringas tríplices e das saídas dos alta rotação sem as peças de mão não estava. A implementação do protocolo para manutenção da qualidade da água dos equipos reduziu a carga bacteriana nas saídas dos alta rotação sem as peças de mão (p=0,004). Enterobactérias e Legionella spp. não foram isoladas de qualquer das amostras de água das torneiras e dos equipos odontológicos (AU)