Priscila Gava Mazzola

The efficacy of chemical agents in cleaning and disinfection programs

BackgroundDue to the growing number of outbreaks of infection in hospital nurseries, it becomes essential to set up a sanitation program that indicates that the appropriate chemical agent was chosen for application in the most effective way.MethodFor the purpose of evaluating the efficacy of a chemical agent, the minimum inhibitory concentration (MIC) was reached by the classic method of successive broth dilutions. The reference bacteria utilized were Bacillus subtilis var. globigii ATCC 9372, Bacillus stearothermophilus ATCC 7953, Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923. The strains of Enterobacter cloacae IAL 1976 (Adolfo Lutz Institute), Serratia marcescens IAL 1478 and Acinetobactev calcoaceticus IAL 124 (ATCC 19606), were isolated from material collected from babies involved in outbreaks of infection in hospital nurseries.ResultsThe MIC intervals, which reduced bacteria populations over 08 log10, were: 59 to 156 mg/L of quaternarium ammonium compounds (QACs); 63 to 10000 mg/L of chlorhexidine digluconate; 1375 to 3250 mg/L of glutaraldehyde; 39 to 246 mg/L of formaldehyde; 43750 to 87500 mg/L of isopropanol or ethanol; 1250 to 6250 mg/L of iodine in polyvinyl-pyrolidone complexes, 150 to 4491 mg/L of chlorine-releasing-agents (CRAs); 469 to 2500 mg/L of hydrogen peroxide; and, 2310 to 18500 mg/L of peracetic acid.ConclusionsChlorhexidine showed non inhibitory activity over germinating spores. A. calcoaceticus, was observed to show resistance to the majority of the agents tested, followed by E. cloacae and S. marcescens.

Determination of decimal reduction time (D value) of chemical agents used in hospitals for disinfection purposes

BackgroundPrior to the selection of disinfectants for low, intermediate and high (sterilizing) levels, the decimal reduction time, D-value, for the most common and persistent bacteria identified at a health care facility should be determined.MethodsThe D-value was determined by inoculating 100 mL of disinfecting solution with 1 mL of a bacterial suspension (104 – 105 CFU/mL for vegetative and spore forms). At regular intervals, 1 mL aliquots of this mixture were transferred to 8 mL of growth media containing a neutralizing agent, and incubated at optimal conditions for the microorganism.ResultsThe highest D-values for various bacteria were determined for the following solutions: (i) 0.1% sodium dichloroisocyanurate (pH 7.0) – E. coli and A. calcoaceticus (D = 5.9 min); (ii) sodium hypochlorite (pH 7.0) at 0.025% for B. stearothermophilus (D = 24 min), E. coli and E. cloacae (D = 7.5 min); at 0.05% for B. stearothermophilus (D = 9.4 min) and E. coli (D = 6.1 min) and 0.1% for B. stearothermophilus (D = 3.5 min) and B. subtilis (D = 3.2 min); (iii) 2.0% glutaraldehyde (pH 7.4) – B. stearothermophilus, B. subtilis (D = 25 min) and E. coli (D = 7.1 min); (iv) 0.5% formaldehyde (pH 6.5) – B. subtilis (D = 11.8 min), B. stearothermophilus (D = 10.9 min) and A. calcoaceticus (D = 5.2 min); (v) 2.0% chlorhexidine (pH 6.2) – B. stearothermophilus (D = 9.1 min), and at 0.4% for E. cloacae (D = 8.3 min); (vi) 1.0% Minncare® (peracetic acid and hydrogen peroxide, pH 2.3) – B. stearothermophilus (D = 9.1 min) and E. coli (D = 6.7 min).ConclusionsThe suspension studies were an indication of the disinfectant efficacy on a surface. The data in this study reflect the formulations used and may vary from product to product. The expected effectiveness from the studied formulations showed that the tested agents can be recommended for surface disinfection as stated in present guidelines and emphasizes the importance and need to develop routine and novel programs to evaluate product utility.

Minimal inhibitory concentration (MIC) determination of disinfectant and/or sterilizing agents

Due to the growing number of outbreaks of infection in hospital and nurseries, it becomes essential to set up a sanitation program that indicates that the appropriate chemical agent was chosen for application in the most effective way. Validating the effectiveness of decontamination and disinfection is an important and often challenging task. In order to study and compare the behavior of selected microorganisms, they were submitted to minimal inhibitory concentration (MIC). The MIC intervals, which reduced bacteria populations over 6 log10, were: 59 to 156 mg/L of quaternary ammonium compounds (QACs); 63 to 10000 mg/L of chlorhexidine; 1375 to 3250 mg/L of glutaraldehyde; 39 to 246 mg/L of formaldehyde; 43750 to 87500 mg/L of ethanol; 1250 to 6250 mg/L of iodine in polyvinyl-pyrolidone complexes, 150 to 4491 mg/L of chlorine-releasing-agents (CRAs) and 469 to 2500 mg/L of hydrogen peroxide. Chlorhexidine showed non inhibitory activity over germinating spores. A. calcoaceticus showed resistance to the majority of the agents tested, followed by E. cloacae and S. marcescens.

Identification of bacteria in drinking and purified water during the monitoring of a typical water purification system.

BackgroundA typical purification system that provides purified water which meets ionic and organic chemical standards, must be protected from microbial proliferation to minimize cross-contamination for use in cleaning and preparations in pharmaceutical industries and in health environments.MethodologySamples of water were taken directly from the public distribution water tank at twelve different stages of a typical purification system were analyzed for the identification of isolated bacteria. Two miniature kits were used: (i) identification system (api 20 NE, Bio-Mérieux) for non-enteric and non-fermenting gram-negative rods; and (ii) identification system (BBL crystal, Becton and Dickson) for enteric and non-fermenting gram-negative rods. The efficiency of the chemical sanitizers used in the stages of the system, over the isolated and identified bacteria in the sampling water, was evaluated by the minimum inhibitory concentration (MIC) method.ResultsThe 78 isolated colonies were identified as the following bacteria genera: Pseudomonas, Flavobacterium and Acinetobacter. According to the miniature kits used in the identification, there was a prevalence of isolation of P. aeruginosa 32.05%, P. picketti (Ralstonia picketti) 23.08%, P. vesiculares 12.82%,P. diminuta 11.54%, F. aureum 6.42%, P. fluorescens 5.13%, A. lwoffi 2.56%, P. putida 2.56%, P. alcaligenes 1.28%, P. paucimobilis 1.28%, and F. multivorum 1.28%.ConclusionsWe found that research was required for the identification of gram-negative non-fermenting bacteria, which were isolated from drinking water and water purification systems, since Pseudomonas genera represents opportunistic pathogens which disperse and adhere easily to surfaces, forming a biofilm which interferes with the cleaning and disinfection procedures in hospital and industrial environments.

Liquid-liquid extraction of commercial and biosynthesized nisin by aqueous two-phase micellar systems.

Nisin is a natural additive for conservation of food, and can also be used as a therapeutic agent. Nisin inhibits the outgrowth of spores, the growth of a variety of Gram-positive and Gram-negative bacteria. In this paper we present a potentially scalable and cost-effective way to purify commercial and biosynthesized in bioreactor nisin, including simultaneously removal of impurities and contaminants, increasing nisin activity. Aqueous two-phase micellar systems (ATPMS) are considered promising for bioseparation and purification purposes. Triton X-114 was chosen as the as phase-forming surfactant because it is relatively mild to proteins and it also forms two coexisting phases within a convenient temperature range. Nisin activity was determined by the agar diffusion assay utilizing Lactobacillus sake as a sensitive indicator microorganism. Results indicated that nisin partitions preferentially to the micelle rich-phase, despite the surfactant concentration tested, and its antimicrobial activity increases. The successful implementation of this peptide partitioning, from a suspension containing other compounds, represents an important step towards developing a separation method for nisin, and more generally, for other biomolecules of interest.

LPS removal from an E. coli fermentation broth using aqueous two-phase micellar system

In biotechnology, endotoxin (LPS) removal from recombinant proteins is a critical and challenging step in the preparation of injectable therapeutics, as endotoxin is a natural component of bacterial expression systems widely used to manufacture therapeutic proteins. The viability of large‐scale industrial production of recombinant biomolecules of pharmaceutical interest significantly depends on the separation and purification techniques used. The aim of this work was to evaluate the use of aqueous two‐phase micellar system (ATPMS) for endotoxin removal from preparations containing recombinant proteins of pharmaceutical interest, such as green fluorescent protein (GFPuv). Partition assays were carried out initially using pure LPS, and afterwards in the presence of E. coli cell lysate. The ATPMS technology proved to be effective in GFPuv recovery, preferentially into the micelle‐poor phase (KGFPuv < 1.00), and LPS removal into the micelle‐rich phase (%REMLPS > 98.00%). Therefore, this system can be exploited as the first step for purification in biotechnology processes for removal of higher LPS concentrations.

Different types of aqueous two‐phase systems for biomolecule and bioparticle extraction and purification

Upstream improvements have led to significant advances in the productivity of biomolecules and bioparticles. Today, downstream processes are the bottleneck in the production of some biopharmaceuticals, a change from previous years. Current purification platforms will reach their physical limits at some point, indicating the need for new approaches. This article reviews an alternative method to extract and purify biomolecules/bioparticles named aqueous two‐phase system (ATPS). Biocompatibility and readiness to scale up are some of the ATPS characteristics. We also discuss some of ATPS applications in the biotechnology field.

Stability of green fluorescent protein (GFP) in chlorine solutions of varying pH.

Green fluorescent protein (GFP) is an excellent biosensor as a result of its ability to be easily monitored in a wide variety of applications. Enzymes and proteins have been used as biological indicators to evaluate the immediate efficacy of industrial procedures, such as blanching, pasteurization, and disinfection treatments, as well as to monitor the satisfactory preservation of a product subjected to disinfection or sterilization. The purpose of this work was to study GFP stability in chlorinated water for injection (WFI) and chlorinated buffered solutions at various pH ranges, with and without agitation, to evaluate the exposure time required for chlorine to decrease 90% of its fluorescence intensity (decimal reduction time, D‐value, min, 25 °C). Fluorescence intensity (Ex/Emmax = 394/509 nm) was measured immediately after the addition of GFP (8.0–9.0 μg/mL) into buffered or unbuffered chlorine solutions with or without constant stirring. With solutions constantly stirred, GFP fluorescence decreased abruptly on contact with chlorine in concentrations greater than 150 ppm, with D‐values between 1.3 min (147 ppm chlorine) and 1.7 min (183 ppm chlorine). In phosphate buffered chlorine solutions (pH = 7.15 ± 0.08), GFP retained its structure between 52 and 94 ppm, but protein stability decreased 10‐fold when exposed to 110 ppm chlorine. The recovery of GFP fluorescence intensity due to renaturation was observed between 30 and 100 ppm chlorine in WFI (final pH = 11.01 ± 0.23) without stirring. Stirring enhanced the contact between GFP and chlorine throughout the assay and provided a more accurate D‐value evaluation. GFP performed as a suitable fluorescent marker for monitoring disinfection effectiveness.

Therapeutic l-asparaginase: upstream, downstream and beyond

Abstract l-asparaginase (l-asparagine amino hydrolase, E.C.3.5.1.1) is an enzyme clinically accepted as an antitumor agent to treat acute lymphoblastic leukemia and lymphosarcoma. It catalyzes l-asparagine (Asn) hydrolysis to l-aspartate and ammonia, and Asn effective depletion results in cytotoxicity to leukemic cells. Microbial l-asparaginase (ASNase) production has attracted considerable attention owing to its cost effectiveness and eco-friendliness. The focus of this review is to provide a thorough review on microbial ASNase production, with special emphasis to microbial producers, conditions of enzyme production, protein engineering, downstream processes, biochemical characteristics, enzyme stability, bioavailability, toxicity and allergy potential. Some issues are also highlighted that will have to be addressed to achieve better therapeutic results and less side effects of ASNase use in cancer treatment: (a) search for new sources of this enzyme to increase its availability as a drug; (b) production of new ASNases with improved pharmacodynamics, pharmacokinetics and toxicological profiles, and (c) improvement of ASNase production by recombinant microorganisms. In this regard, rational protein engineering, directed mutagenesis, metabolic flux analysis and optimization of purification protocols are expected to play a paramount role in the near future.

Stability, purification, and applications of bromelain: A review.

Bromelain is a cysteine protease found in pineapple tissue. Because of its anti‐inflammatory and anti‐cancer activities, as well as its ability to induce apoptotic cell death, bromelain has proved useful in several therapeutic areas. The market for this protease is growing, and several studies exploring various properties of this molecule have been reported. This review aims to compile this data, and summarize the main findings on bromelain in the literature to date. The physicochemical properties and stability of bromelain under different conditions are discussed. Several studies on the purification of bromelain from crude extracts using a wide range of techniques such as liquid–liquid extractions by aqueous two‐phase system, ultrafiltration, precipitation, and chromatography, have been reported. Finally, the various applications of bromelain are presented. This review therefore covers the main properties of bromelain, aiming to provide an up‐to‐date compilation of the data reported on this enzyme.