Microbiological validation of a robot for the sterile compounding of injectable non-hazardous medications in a hospital environment.

Objectives: To design and execute a comprehensive microbiological validation protocol to assess a brand-new sterile compounding robot in a hospital pharmacy environment, according to ISO and EU GMP standards. Methods: Qualification of the Class-A inner environment of the robot was performed through microbial air and surface quality assessment utilising contact plates, swabs and particulate matter monitoring. To evaluate the effectiveness of the microbial decontamination process (UV rays) challenge test against Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis spores and Candida albicans was used. The challenge Media Fill test was used to validate the aseptic processing. Results: After 3 hours, no microorganisms retained viability. Monitoring inside the equipment evidenced complete absence of microorganisms. The Media Fill test was always negative. Conclusions: According to our results, the APOTECAunit meets the requirements for advanced aseptic processing in the hospital pharmacies and the pharmaceutical industry in general, providing advantages in terms of safety for patients compared with conventional procedures of parenteral preparation production. The protocol has demonstrated to be a comprehensive and valuable tool in validating, from a microbial point of view, a sterile-compounding technology. This study might represent an important benchmark in developing a contamination control strategy, as required, for example, in the Performance Qualification of the GMP in the case of drug manufacturing.

Interactions of Nitroxide-Conjugated and Non-Conjugated Glycodendrimers with Normal and Cancer Cells and Biocompatibility Studies.

Poly(propyleneimine) glycodendrimers fully modified with maltose units were administered to different cancer cell lines and their effect on cell viability was evaluated by using MTS assay and flow cytometry. The mechanism of dendrimer-cell interactions was investigated by the electron paramagnetic resonance (EPR) technique by using a new nitroxide-conjugated glycodendrimer. The nitroxide groups did not modify both the biological properties (cell viability and apoptosis degree) of the dendrimers in the presence of the cells and the dendrimer-cell interactions. Since this class of dendrimers is already known to be biocompatible for human healthy cells, noncancer cells such as human peripheral blood mononuclear cells (PBMCs) and macrophages were also treated with the glycodendrimer, and EPR spectra of the nitroxide-conjugated glycodendrimer were compared for cancer and noncancer cells. It was found that this dendrimer selectively affects the cell viability of tumor cells, while, surprisingly, PBMC proliferation is induced. Moreover, H-bond-active glycodendrimer-cell interactions were different for the different cancer cell lines and noncancer cells. The nitroxide-conjugated glycodendrimer was able to interact with the cell membrane and eventually cross it, getting in contact with cytosol antioxidants. This study helps to clarify the potential anticancer effect of this class of dendrimers opening to future applications of these macromolecules as new antitumor agents.

Dendrimers and Polyamino-Phenolic Ligands: Activity of New Molecules Against Legionella pneumophila Biofilms.

Legionnaires' disease is a potentially fatal pneumonia caused by Legionella pneumophila, an aquatic bacterium often found within the biofilm niche. In man-made water systems microbial biofilms increase the resistance of legionella to disinfection, posing a significant threat to public health. Disinfection methods currently used in water systems have been shown to be ineffective against legionella over the long-term, allowing recolonization by the biofilm-protected microorganisms. In this study, the anti-biofilm activity of previously fabricated polyamino-phenolic ligands and polyamidoamine dendrimers was investigated against legionella mono-species and multi-species biofilms formed by L. pneumophila in association with other bacteria that can be found in tap water (Aeromonas hydrophila, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae). Bacterial ability to form biofilms was verified using a crystal violet colorimetric assay and testing cell viability by real-time quantitative PCR and Plate Count assay. The concentration of the chemicals tested as anti-biofilm agents was chosen based on cytotoxicity assays: the highest non-cytotoxic chemical concentration was used for biofilm inhibition assays, with dendrimer concentration 10-fold higher than polyamino-phenolic ligands. While Macrophen and Double Macrophen were the most active substances among polyamino-phenolic ligands, dendrimers were overall twofold more effective than all other compounds with a reduction up to 85 and 73% of legionella and multi-species biofilms, respectively. Chemical interaction with matrix molecules is hypothesized, based on SEM images and considering the low or absent anti-microbial activity on planktonic bacteria showed by flow cytometry. These data suggest that the studied compounds, especially dendrimers, could be considered as novel molecules in the design of research projects aimed at the development of efficacious anti-biofilm disinfection treatments of water systems in order to minimize legionellosis outbreaks.

Evaluation of ultraviolet irradiation efficacy in an automated system for the aseptic compounding using challenge test.

OBJECTIVE: Ultraviolet (UV) irradiation efficacy in the intravenous compounding robot APOTECAchemo was evaluated to define the best operative conditions in terms of sterility and time optimization. DESIGN: The challenge test was used against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Bacillus subtilis spores and Candida albicans. Inoculated plates were placed inside the robot and irradiated for different times. Microbial air and surface quality inside the equipment were monitored utilizing settle and contact plates, swabs. RESULTS: After 4 h, no microorganisms were viable with killing rates ranging from 5- to 7-log for different microorganisms after 1 h of exposition. In confirmation of the efficacy of the UV irradiation program adopted, the microbial monitoring inside the equipment always gave negative results. CONCLUSIONS: This is the first exhaustive investigation of UV irradiation efficacy in the aseptic pharmaceutical production. We demonstrated that UV irradiation plays an essential role in maintaining the sterility condition of the workplace inside the APOTECAchemo and assuring the standards for aseptic manufacturing of medicinal drugs, as required for Class A clean areas. A 4-h UV irradiation also ensures sterility in the case of very resistant microorganisms and in the presence of high microbial charge (10(8) CFU/ml), but a killing rate of 5 or more is already recorded after the first hour of exposition. The results provide useful information for the best operative conditions in terms of both sterility and time optimization, not only for the automated compounding, but also for the traditional aseptic manufacturing processes.

Evaluation of Escherichia coli viability by flow cytometry: A method for determining bacterial responses to antibiotic exposure.

BACKGROUND: In this study, we check for the presence of specific resistance genes by polymerase chain reaction (PCR) and then we used flow cytometry (FCM) to evaluate antibiotic-induced effects in different strains of Escherichia coli (E. coli). METHODS: The presence of resistance genes was investigated by PCR in 10 strains of E. coli isolated from Foglia River. Bacterial responses to different antibiotics were also tested with FCM techniques by evaluating both the degree of decrease in viability and the light scatter changes in all of the strains. RESULTS: PCR revealed that only one strain exhibits the presence of one resistance gene. Despite this, analyses of strains using FCM evidenced the presence of viable subpopulations after antibiotic treatment. Furthermore, analyses of scatter signals revealed profound changes in the Forward Scatter and Side Scatter of the bacterial populations as a consequence of antibiotic exposure, confirming the viability and membrane potential data. The riverine strains were in general less sensitive to antibiotics than the reference strain (ATCC 25922). CONCLUSIONS: Antibiotic resistance is a widespread phenomena. The multiparametric approach based on FCM used in this study, providing results about different aspects (cell viability, membrane potential, light scatter changes), may overcome the limitation of PCR and could represent an adequate method for the evaluation of bacteria responses to antibiotic exposure.

A multi-approach study of influence of growth temperature and nutrient deprivation in a strain of Aeromonas hydrophila.

In the present study we investigated the behavior of an Aeromonas hydrophila strain in prolonged nutrient deprivation condition analyzing the possible link among survival, cell morphology and adhesive characteristics and correlating them with the expression of the 43kDa outer membrane protein (OMP). The strain was inoculated in mineral and drinking chlorinated water, and in Nutrient Broth as a control with incubation at 4 and 24°C for 176days. Specimens were analyzed at different times during starvation stress. Viability was assessed by flow cytometry and growth by plate count technique; morphology and adhesivity were detected by optical and electron microscopy. The 43kDa OMP expression at different times was determined after immunoblotting assay using a polyclonal antibody produced in rabbit. The results showed a long-term viability as evidenced by cytofluorimetric analysis; however, the prolonged starvation led to the shift from the normal rod shaped cells to spherical forms in the last phases of incubation especially at 24°C. Concomitantly with the appearance of spherical cells we noted a reduction of the 43kDa OMP content and adhesive ability. Therefore, our results suggest a role of the 43kDa OMP as adhesin in A. hydrophila. In conclusion, we demonstrated that the bacterium can long survive under stress conditions, however adopting strategies which can lead to a loss of some cell surface components involved in the interactions with eukaryotic cells, therefore modifying its virulence properties.

Evaluation of Escherichia coli viability by flow cytometry; a method for determining bacterial responses to antibiotic exposure.

BACKGROUND: In this study, we check for the presence of specific resistance genes by PCR and then we used flow cytometry to evaluate antibiotic-induced effects in different strains of Escherichia coli. METHODS: The presence of resistance genes was investigated by PCR in 10 strains of Escherichia coli isolated from Foglia River. Bacterial responses to different antibiotics were also tested with flow cytometry techniques by evaluating both the degree of decrease in viability and the light scatter changes in all of the strains. RESULTS: PCR revealed that only one strain exhibits the presence of one resistance gene. Despite this, analyses of strains using flow cytometry evidenced the presence of viable subpopulations after antibiotic treatment. Furthermore, analyses of scatter signals revealed profound changes in the Forward Scatter (FSC) and Side Scatter (SSC) of the bacterial populations as a consequence of antibiotic exposure, confirming the viability and membrane potential data. The riverine strains were in general less sensitive to antibiotics than the reference strain (ATCC 25922). CONCLUSIONS: Antibiotic resistance is a widespread phenomena. The multiparametric approach based on flow cytometry used in this study, providing results about different aspects (cell viability, membrane potential, light scatter changes), may overcome the limitation of PCR and could represent an adequate method for the evaluation of bacteria responses to antibiotic exposure. This article is protected by copyright. All rights reserved.

Chemical and microbiological monitoring of air in two waste incineration plants.

The study evaluated emissions from two incinerator plants and occupational exposure of workers during a six-year monitoring period (2004-2009). Chemical and microbiological analyses were performed by collecting environmental and staff individual air samples. Inspirable and respirable particulate and metals were measured in both environmental and staff samples; concentrations of Volatile Organic Compounds and the presence of microorganisms were investigated only in environmental samples. The concentrations of all organic and inorganic compounds investigated were very low and always below the Italian legal limits and the threshold limits recommended by the American Conference of Governmental Industrial Hygienists. In addition, microbial contamination was generally low in all the working areas considered. Study results revealed a good quality of air in the incineration plants and no apparent risk situation for the workers.

A severe case of Aeromonas veronii biovar sobria travellers' diarrhoea characterized by Vibrio parahaemolyticus co-isolation.

We report a severe case of travellers' diarrhoea in a patient returning from Ecuador to Italy with the concomitant presence of Aeromonas veronii biovar sobria and Vibrio parahaemolyticus in their faeces. Based on diagnostic results, epidemiological information and the clinical outcome, we conclude that the real aetiological agent was A. veronii biovar sobria, while V. parahaemolyticus was only transient in the intestine of the patient.

Isolation of a strain of Aspergillus fumigatus able to grow in minimal medium added with an industrial cyanide waste.

The present note refers the results about the isolation of an Aspergillus fumigatus strain able to grow on an industrial cyanide waste as nitrogen source. The fungus was selected from an alkaline unpolluted soil in enrichment cultures in 50 ml of Minimal Medium added with 20 mmol glucose and supplemented initially with 0.1 mmol KCN and then with 70 µl of a waste solution from a jewelry industry containing free cyanide and cyanide complexes of heavy metal ions including copper, silver, nickel, and others. The cyanide content of the waste was 1,500 ppm. The fungal growth was monitored determining dry weight, protein content and glucose consumption. The fungus efficiently utilized the cyanide as evidenced by the decrease in the inoculated medium of the compound under detection limits within 24 h and the concomitant growth within 15 days during which periodical additions of the waste to the cultures were made. The amount of the cyanide in the biomass of the fungus grown in presence of the waste was very scarce and comparable to that in absence of the pollutant. Furthermore the fungus was able to sequestrate metals such Ag, Cu, and Ni as a resistance mechanism against heavy metals. In conclusion our results are of interest for biodegradation plans of electroplating industrial wastes containing cyanide based pollutants.

Unusual case of necrotizing fasciitis caused by Vibrio cholerae O137.

We report a case of necrotizing fasciitis caused by Vibrio cholerae O137 in an immunocompromised 49-year-old man. The infection was acquired following a minor traumatic injury and exposure to seawater during the summer of 2009 in Italy. Although highly immunocompromised, the patient survived. The strain was cytotoxic, invasive, and adhesive and contained a fragment of the El Tor-like hemolysin (El Tor hlyA) gene.

Putative virulence properties of Aeromonas strains isolated from food, environmental and clinical sources in Italy: a comparative study.

The distribution of virulence properties in 142 strains of Aeromonas isolated from diarrhoeic patients, food and surface water in Italy and identified by biochemical and molecular methods was investigated. The virulence properties studied were the presence of genes for the aerolysin (aerA), heat-stable cytotonic enterotoxin (ast), heat-labile cytotonic enterotoxin (alt), cytotoxic enterotoxin (act); and cytotoxicity for Vero cells and adhesion on Hep-2 cells. A. hydrophila and A. caviae were the species most commonly isolated from clinical and environmental samples (9/30; 30.0% and 5/27; 18.5%, respectively) while mesophilic A. salmonicida was most common in food samples (19/80; 23.7%). Out of 142 strains, 86 (60.6%) were positive for at least one of the virulence properties. All the toxin genes were present in 4/18 (22.3%) of clinical strains. Most of the food isolates (54/55; 98.2%) were cytotoxic and most of the environmental strains (12/13; 92.3%) were adhesive. The aerA gene was present in most toxigenic strains (72/86; 83.7%), irrespective of their origin. The growth temperature affected the expression of cytotoxicity and adhesivity. Aeromonas strains from food and surface water frequently had toxin gene patterns similar to those of clinical strains and expressed virulence properties at human body temperature. These findings indicate that aeromonads have the potential to cause human illness and confirm the role of food and water as vehicles for Aeromonas diseases.

Prevalence and virulence properties of non-O1 non-O139 Vibrio cholerae strains from seafood and clinical samples collected in Italy.

Seafood and clinical samples collected in Italy during 2006 were analyzed to evaluate prevalence, serological and virulence properties of non-O1 non-O139 Vibrio cholerae (NCV) isolates. Biochemical and serological characterization of the strains was performed by standardized procedures while virulence properties of NCVs were assayed by molecular, in vivo and in vitro toxicological methods. Of the 300 seafood samples examined, including mussel, cod, mackerel, anchovy, clam, prawn and cuttlefish, 5.6% were positive for NCVs: 4.7% and 8.5% from local and imported seafood, respectively. The prevalence of NCVs was highest in prawn (16.6%) and mussel (7.7%). Of 58 hospitalized patients that presented acute diarrhea, 3.4% eliminated NCVs in stools 24-48 h after consumption of seafood. All NCVs had ToxR and hlyAET genes but lacked ctxA, zot, and tcpA genes. One isolate from prawn had stn/sto gene. All strains were hemolytic, cytotoxic, and able to induce intestinal and extraintestinal effects on the suckling mouse model. Our results confirm that non-toxigenic NCVs that express the gene encoding El Tor-like hemolysin can be isolated from patients suffering a cholera-like syndrome after consumption of seafood. This evidence along with the virulence and enteropathogenicity of all the ctxA(-) tcpA(-) zot(-) stn/sto(-) hlyAET(+) NCV isolates in the experimental model, suggest that El Tor-like hemolysin may play an important role in human pathogenesis. Moreover, the isolates from seafood showed molecular, biological and enzymatic patterns similar to those isolated from clinical samples, underlining that environmental NCVs are potentially able to induce human infections and confirming the important role of seafood as a vehicle of V. cholerae diseases.

Determination of viability of Aeromonas hydrophila in increasing concentrations of sodium chloride at different temperatures by flow cytometry and plate count technique.

Aeromonads in waters and foods can represent a risk to human health. Factors such as sodium chloride concentration and temperature can affect growth and viability of several food and water-borne pathogens. The behaviour of an Aeromonas hydrophila strain in the presence of 1.7%, 3.4% and 6% NaCl concentrations at 24 degrees C and 4 degrees C was studied over a 188 day period. Viability and membrane potential were assessed by flow cytometry; growth was evaluated by plate count technique. Flow cytometry evidenced that A. hydrophila retained viability over the period although varying according to temperature and salt concentrations. Colony Forming Units were generally lower in number than viable cells especially in the presence of 6% NaCl, indicating the occurrence of stressed cells which maintain metabolic activity yet are not able to grow on agar plates. In conclusion, A. hydrophila showed a long-term halotolerance even at elevated (6%) NaCl concentrations and a lesser sensitivity to salt at low temperature; therefore, low temperature and salt, which are two important factors limiting bacterial growth, do not assure safety in the case of high initial contamination. Finally, cytometry appears a valid tool for the rapid detection of the viability of pathogenic bacteria in food and environmental matrices to control and prevent health risks.

Bacterial cell monitoring in wastewater treatment plants by flow cytometry.

The activated sludge process is performed by a variable and mixed community of microorganisms in an aerobic aquatic environment, in which bacteria constitute the majority and represent the main microorganisms responsible for the degradation process in a plant. In this work, we monitored bacterial charge in different wastewater treatment plants by flow cytometry, also evaluating chlorination effects on bacterial viability, both by flow cytometry and traditional plate counts. Maximum values of bacterial charge were registered in the aeration tank of all plants monitored. Cell viability did not show significant differences (p > 0.05) in samples collected in "before chlorination" and "wastewater effluent" treatment steps; this suggests that the chlorination was not able to decrease total viable bacterial charge. In this work, we discuss the need to improve microbiological analyses, both in terms of measuring other potential pathogens and of using new methodological approaches in the traditional evaluation of the microbiological quality of effluents.

Inactivation of Legionella pneumophila by combined systems of copper and silver ions and free chlorine.

The aim of this study was to evaluate the efficacy of copper and silver ions and of free chlorine in different combinations and concentrations (0.4 to 0.8-0.04 to 0.08 mg/l Cu(2+) Ag(+); 0.4 to 0.8-0.04 to 0.08-0.2 mg/l Cu(2+) Ag(+) Cl; 0.4 to 0.8-0.04 to 0.08-2 mg/l Cu(2+) Ag(+) Cl; 0.4 to 0.8-0.04 to 0.08-4 mg/l Cu(2+) Ag(+) Cl; 2-20 mg/l Cl), in inactivating Legionella pneumophila in drinking and distilled water after a contact time of 24-hours. Treatment with chlorine alone at 20 mg/l concentration was found to be the most effective treatment leading to complete killing of bacteria within 4 minutes in all water samples. On the other hand, at 2 mg/l concentration complete inactivation was obtained after 3 hours. The association of copper and silver ions at concentrations of 0.4-0.04 mg/l was found to be less effective and live bacteria could still be identified in all water samples after a 24 hour contact time. When testing copper and silver ions in combination, at concentrations of 0.8-0.08 mg/l and different combinations of the three disinfectants, results varied according to the various concentrations and type of water. The combination of copper and silver with 2 mg/l of chlorine was found to be more effective than 2mg/l of chlorine alone; a synergistic effect can therefore be hypothesized. The physical and chemical properties of drinking water, in particular its chlorine content, may have affected the water disinfection process when disinfecting agents were used in low concentrations. In conclusion, this study confirms the efficacy of shock hyperchlorination in the inactivation of Legionella pneumophila. However, the combination of free chlorine with metal (copper and silver) ions may represent a valid option for reducing the concentration of disinfectants to safer levels for human health and avoiding damage to water distribution systems especially in facilities such as hotels and hospitals.

Techniques for the recovery of enteric viruses from artificially contaminated marine sediments.

Viruses are an important component of aquatic microbial communities and marine sediments may represent an optimal means for their survival. The aim of this study was to evaluate different methods for virus recovery from marine sediments. Three methods were used for virus recovery from artificially contaminated sediments: elution and centrifugation technique, sonication technique, and mechanical disgregation followed by elution and centrifuge technique. The sonication technique obtained the highest virus recovery percentages (94,25%). Eluent 2 provided more efficient recovery of enteric viruses than eluent 1 presumably due to the presence, in eluent 2, of NANO3, a chaotropic agent that enhances the solubilization of hydrophobic compounds in water. Finally, the authors confirm the importance of searching for viruses in sediments, which protect them from inactivation by biological, chemical and physical factors and allow them to survive longer than in the overlaying water column.

Influence of different concentrations of nitrogen and phosphorous on Aeromonas spp. growth.

This paper evaluates the influence of nitrogen and phosphorous on the growth of Aeromonas previously isolated from irrigation water. Three strains respectively of three species of Aeromonas (A. hydrophila, A. caviae, A. sobria) (nine strains) were individually inoculated in a microcosm added with ammonium, nitrate and phosphate using different combinations (NH4-N + NO3-N and NH4-N + NO3-N + PO4-P) and concentrations (NH4-N: from 20 to 2500 microg l-1; NO3-N: from 200 to 22000 microg l-1; PO4-P: from 20 to 1200 microg l-1). A positive correlation was found between bacterial growth and NH4-N charge, but not a strict correlation with NO3-N. The addition of PO4-P at concentrations from 20 to 200 microg l-1 stimulated the bacterial growth; inversely, concentrations of 500 and 1200 microg l-1 were inhibitory. Ammonium, nitrate and phosphate influence in different way Aeromonas growth; so, their various concentrations can partially explain the different recovery of these bacteria from aquatic environments. There is discordance in literature about the influence of environmental parameters on Aeromonas growth in aquatic ecosystems. Therefore, this study contributes to a better understanding about interactions of some chemical parameters and Aeromonas growth, which are at the basis of the varied relationship described for environmental factors governing aeromonads densities.

Determination of the viability of Aeromonas hydrophila in different types of water by flow cytometry, and comparison with classical methods.

The presence of Aeromonas spp. in water can represent a risk for human health. Therefore, it is important to know the physiological status of these bacteria and their survival in the environment. We studied the behavior of a strain of Aeromonas hydrophila in river water, spring water, brackish water, mineral water, and chlorinated drinking water, which had different physical and chemical characteristics. The bacterial content was evaluated by spectrophotometric and plate count techniques. Flow cytometric determination of viability was carried out using a dual-staining technique that enabled us to distinguish viable bacteria from damaged and membrane-compromised bacteria. The traditional methods showed that the bacterial content was variable and dependent on the type of water. The results obtained from the plate count analysis correlated with the absorbance data. In contrast, the flow cytometric analysis results did not correlate with the results obtained by traditional methods; in fact, this technique showed that there were viable cells even when the optical density was low or no longer detectable and there was no plate count value. According to our results, flow cytometry is a suitable method for assessing the viability of bacteria in water samples. Furthermore, it permits fast detection of bacteria that are in a viable but nonculturable state, which are not detectable by conventional methods.

Isolation of Escherichia coli O157 from human and bovine faeces in the Urbino area, Italy.

We examined 476 faecal samples from subjects aged from 0 to >60 years, 283 with diarrhoea and 193 with illnesses involving other sites or clinically healthy, and 154 samples of faeces of healthy cattle, in order to define the diffusion of E. coli O157 in the Urbino area. The samples were seeded by both direct streaking onto cefixime tellurite sorbitol Mac Conkey agar (CT-SMAC) and previous enrichment in cefixime tellurite tryptone soya broth for human specimens and in cefixime vancomicin tryptone soya broth for bovine samples. The strains of E. coli O157 were characterized by verocytotoxin and adhesin eae genes detection. We isolated one strain of E. coli O157 (0.2%) from a man 68 year old who had bloody diarrhoea, and one strain (0.64%) from a weaned calf. Both isolates carried the adhesin eae gene, but only the bovine strain was VT2+. The study shows a low diffusion of E. coli O157 in the Urbino area, confirming the epidemiological data on the national territory.