A Pilot Study for Legionella pneumophila Volatilome Characterization Using a Gas Sensor Array and GC/MS Techniques.

Legionellosis is a generic term describing the pneumonic (Legionnaires' disease, LD) and non-pneumonic (Pontiac fever, PF) forms of infection with bacteria belonging to the genus Legionella. Currently, the techniques used to detect Legionella spp. in water samples have certain limitations and drawbacks, and thus, there is a need to identify new tools to carry out low-cost and rapid analysis. In this regard, several studies demonstrated that a volatolomics approach rapidly detects and discriminates different species of microorganisms via their volatile signature. In this paper, the volatile organic compounds (VOCs) pattern emitted in vitro by Legionella pneumophila cultures is characterized and compared to those produced by other Legionella species and by Pseudomonas aeruginosa, using a gas sensor array and gas chromatograph mass spectrometer (GC-MS). Bacterial cultures were measured at the 3rd and 7th day after the incubation. Sensor array data analyzed via the K-nearest neighbours (k-NN) algorithm showed a sensitivity to Legionella pneumophila identification at around 89%. On the other hand, GC-MS identified a bouquet of VOCs, mainly alcohols and ketones, that enable the differentiation of Legionella pneumophila in respect to other waterborne microorganisms.

Mapping Biological Risks Related to Necropsy Activities: Old Concerns and Novel Issues for the Safety of Health Professionals.

Nowadays only a few studies on biological and environmental risk among healthcare workers are available in literature. The present study aims to assess the health operator's risk of contact with microorganisms during necropsy activities, to evaluate the efficiency of current protections, to identify possible new sources of contact, and to point out possible preventive measures. In addition, considering the current pandemic scenario, the risk of transmission of SARS-CoV-2 infection in the dissection room is assessed. The objectives were pursued through two distinct monitoring campaigns carried out in different periods through sampling performed both on the corpses and at the environmental level.

Phylogenomic analysis and characterization of carbon monoxide utilization genes in the family Phyllobacteriaceae with reclassification of Aminobacter carboxidus (Meyer et al. 1993, Hördt et al. 2020) as Aminobacter lissarensis comb. nov. (McDonald et al. 2005).

The monotypic carboxydophilic genus Carbophilus has recently been transferred to the genus Aminobacter within the family Phyllobacteriaceae, and Carbophilus carboxidus was renamed Aminobacter carboxidus (comb. nov.) [Hördt et al. 2020]. Due to the poor resolution of the 16S rRNA gene-based phylogeny, an extensive phylogenomic analysis of the family Phyllobacteriaceae was conducted, with particular focus on the genus Aminobacter. Whole genome-based analyses of Phyllobacteriaceae type strains provided evidenced that the genus Aminobacter forms a monophyletic cluster, clearly demarcated from all other members of the family. Close relatedness between A. carboxidus DSM 1086T and A. lissarensis DSM 17454T was inferred from core proteome phylogeny, shared gene content, and multilocus sequence analyses. ANI and GGDC provided genetic similarity values above the species demarcating threshold for these two type strains. Metabolic profiling and cell morphology analysis corroborated the phenotypic identity between A. carboxidus DSM 1086T and A. lissarensis DSM 17454T. Search for the presence of carbon monoxide dehydrogenase (CODH) genes in Phyllobacteriaceae genomes revealed that the form II CODH is widespread in the family, whereas form I CODH was detected in few Mesorhizobium type strains, and in both A. carboxidus DSM 1086T and A. lissarensis DSM 17454T. Results of phylogenomic, chemotaxonomic, and morphological investigations, combined with the presence of similarly arranged CODH genes, indicate that A. carboxidus DSM 1086T and A. lissarensis DSM 17454T are distinct strains of the same species. Hence A. carboxidus is a later subjective heterotypic synonym of A. lissarensis.

Aspergillus Species Discrimination Using a Gas Sensor Array.

The efficiency of electronic noses in detecting and identifying microorganisms has been proven by several studies. Since volatile compounds change with the growth of colonies, the identification of strains is highly dependent on the growing conditions. In this paper, the effects of growth were investigated with different species of Aspergillus, which is one of the most studied microorganisms because of its implications in environmental and food safety. For this purpose, we used an electronic nose previously utilized for volatilome detection applications and based on eight porphyrins-functionalized quartz microbalances. The volatile organic compounds (VOCs) released by cultured fungi were measured at 3, 5, and 10 days after the incubation. The signals from the sensors showed that the pattern of VOCs evolve with time. In particular, the separation between the three studied strains progressively decreases with time. The three strains could still be identified despite the influence of culture time. Linear Discriminant Analysis (LDA) showed an overall accuracy of 88% and 71% in the training and test sets, respectively. These results indicate that the presence of microorganisms is detectable with respect to background, however, the difference between the strains changes with the incubation time.

The relevance of molecular genotyping to allocate cases in a suspected outbreak of Legionella pneumonia in patients with prolonged immunosuppressive therapy.

Three cases of pneumonia caused by Legionella pneumophila serogroup 1 (Lp1) in immunosuppressed patients with repeated hospitalization were suspected as a healthcare-associated cluster. The environmental investigation did not reveal the presence of legionellae in the hospital patient rooms. Water samples collected from the homes of two patients were also negative for Legionella spp. In the absence of environmental strains potentially involved in the infections, we proceeded to genotype environmental Lp1 strains isolated in the hospital during routine water sampling during the decade 2009-2019 and recovered after long-term storage at -20°C. These 'historical' strains exhibited a high grade of similarity and stability over time, regardless of the disinfection systems. The different molecular profiles shown among the clinical and environmental strains excluded a nosocomial outbreak. The study suggests that the application of molecular typing may be a useful tool to discriminate hospital vs community-acquired cases, mostly for severely immunosuppressed patients in whom the symptomatology could be insidious and the incubation period could be prolonged. Moreover, the genotyping allowed us to exclude any link between the cases.

Nanomaterials as a new opportunity for protecting workers from biological risk.

Healthcare-Associated Infections (HAIs) represent a frequent complication for hospitalized patients and more rarely for workers. In recent years, substantial scientific evidence has been reached regarding the role played by the inanimate surfaces, especially those touched in patient-care areas, in the transmission of nosocomial pathogens. Therefore, it is essential to find new collective protective measures to minimize microbial contamination in healthcare facilities, thereby preventing the spread of multi-drug resistant bacteria. We present an overview of the major nano-enabled AntiMicrobial Coatings (AMCs) which may be used as collective protective measures in healthcare setting, discussing also some aspects related to their effectiveness and safety. AMCs may be classified within three groups on base of their mechanism of action: surfaces releasing active compound, contact-killing surfaces and anti-adhesive surfaces. To date, little information is available on the effectiveness of AMCs to reduce the risk of HAIs since the most of studies do not reach conclusive results on their beneficial effects. Moreover, the lack of standard protocols for assessing antimicrobial efficacy and poor data about the interaction between AMCs and disinfectants prevent their placing on the market. Further studies are needed for assessing risks and benefits of AMCs as collective protective measures in healthcare setting.

Susceptibility biomarker detection in urine exfoliate DNA.

AIM: The occupational biomonitoring of exposures to carcinogens is carried out by measuring dose (metabolites) and susceptibility biomarkers (gene polymorphisms) in two biological matrices: urine for metabolite detection and blood for genotyping. Blood is the most common substrate but has some disadvantages including: invasiveness of the harvesting technique; need of specialized staff and equipment; and high infection risk. METHODS & RESULTS: We propose our in-house approach using urine as single sample in 20 volunteers for simultaneous detection of dose and susceptibility biomarkers in order to verify efficacy and feasibility. CONCLUSION: Despite the low number of subjects, interindividual and gender variability in DNA yield, urine genomic DNA is a valuable source for gene polymorphism studies when blood samples are not available. [Formula: see text].

Control of Legionella Contamination and Risk of Corrosion in Hospital Water Networks following Various Disinfection Procedures.

Physical and chemical disinfection methods have been proposed with the aim of controlling Legionella water contamination. To date, the most effective procedures for reducing bacterial contamination have not yet been defined. The aim of this study was to assess the long-term effectiveness of various disinfection procedures in order to reduce both culturable and nonculturable (NC) legionellae in different hospital water networks treated with heat, chlorine dioxide, monochloramine, and hydrogen peroxide. The temperature levels and biocide concentrations that proved to give reliable results were analyzed. In order to study the possible effects on the water pipes, we verified the extent of corrosion on experimental coupons after applying each method for 6 months. The percentage of positive points was at its lowest after treatment with monochloramine, followed by chlorine dioxide, hydrogen peroxide, and hyperthermia. Different selections of Legionella spp. were observed, as networks treated with chlorine-based disinfectants were contaminated mainly by Legionella pneumophila serogroup 1, hyperthermia was associated with serogroups 2 to 14, and hydrogen peroxide treatment was associated mainly with non-pneumophila species. NC cells were detected only in heat-treated waters, and also when the temperature was approximately 60°C. The corrosion rates of the coupons were within a satisfactory limit for water networks, but the morphologies differed. We confirm here that chemical disinfection controls Legionella colonization more effectively than hyperthermia does. Monochloramine was the most effective treatment, while hydrogen peroxide may be a promising alternative to chlorine-based disinfectants due to its ability to select for other, less virulent or nonpathogenic species.

Biomarkers of susceptibility following benzene exposure: influence of genetic polymorphisms on benzene metabolism and health effects.

Benzene is a ubiquitous occupational and environmental pollutant. Improved industrial hygiene allowed airborne concentrations close to the environmental context (1-1000 µg/m(3)). Conversely, new limits for benzene levels in urban air were set (5 µg/m(3)). The biomonitoring of exposure to such low benzene concentrations are performed measuring specific and sensitive biomarkers such as S-phenylmercapturic acid, trans, trans-muconic acid and urinary benzene: many studies referred high variability in the levels of these biomarkers, suggesting the involvement of polymorphic metabolic genes in the individual susceptibility to benzene toxicity. We reviewed the influence of metabolic polymorphisms on the biomarkers levels of benzene exposure and effect, in order to understand the real impact of benzene exposure on subjects with increased susceptibility.

Influence of genetic polymorphisms of styrene-metabolizing enzymes on the levels of urinary biomarkers of styrene exposure.

Styrene exposure is still present in different occupational settings including manufacture of synthetic rubber, resins, polyesters and plastic. The aim of this work was to investigate the effects of polymorphic genes CYP2E1, EPHX1, GSTT1, and GSTM1 on the urinary concentrations of the styrene metabolites mandelic acid (MA), phenylglyoxylic acid (PGA) and on the concentration ratios between (MA+PGA) and urinary styrene (U-Sty) and airborne styrene (A-Sty), in 30 workers from two fiberglass-reinforced plastic manufacturing plants and 26 unexposed controls. Personal air sampling and biological monitoring results revealed that sometimes exposure levels exceeded both the threshold limit value (TLV) and the biological exposure index (BEI) suggested by the American Conference of Governmental Industrial Hygienists. A significantly reduced excretion of styrene metabolites (MA+PGA) in individuals carrying the CYP2E1*5B and CYP2E1*6 heterozygote alleles, with respect to the homozygote wild type, was observed only in the exposed group. A reduction was also detected, in the same group, in subjects carrying the slow allele EPHX1 (codon 113), through the lowering of (MA+PGA)/urinary styrene concentration ratio. In addition, the ratio between MA+PGA and the personal airborne styrene concentration appeared to be modulated by the predicted mEH activity, in the exposed group, as evidenced by univariate linear regression analysis. Our results confirm some previous hypotheses about the role of the polymorphism of genes coding for enzymes involved in the styrene detoxification pathway: this may significantly reduce the levels of excreted metabolites and therefore it must be taken into account in the interpretation of the biological monitoring results for occupational exposure.

Influence of genetic polymorphism on t,t-MA/S-PMA ratio in 301 benzene exposed subjects.

This study investigated the effect of polymorphic genes GSTT1, GSTM1, GSTA1, EHPX1, NQO1, CYP2E1, CYP1A and MPO on the urinary concentrations and ratio (R) of the benzene metabolites trans,trans-muconic acid (t,t-MA) and S-phenyl mercapturic acid (S-PMA) in 301 oil refinery workers. The metabolites' concentrations are lower and R is higher (100.66) in non-smokers (n=184) than in smokers (n=117, R=36.54). Non-smokers have lower S-PMA and a higher R in GSTT1 null genotypes than in positive, and a higher S-PMA and a lower R in GSTA1 wild type genotypes. In smokers the GSTT1 null genotype effect on both S-PMA and R is confirmed, and is also shown in GSTM1 null, but not in GSTA1 wild type genotypes. GSTT1 null polymorphism reduces the conjugation rate of benzene epoxide with GSH, and to a lesser extent also GSTTA1 mutant, GSTM1 null and NQO1 mutant genotypes. The activity of one GST is compensated by another in GSTM1 and GSTA1 defective subjects, but not in GSTT1 null genotypes, whose average S-PMA excretion is about 50% with respect to the positive ones, for the same benzene exposure. R showed to be a more sensitive marker for these effects than the metabolite levels.

Low occupational exposure to benzene in a petrochemical plant: modulating effect of genetic polymorphisms and smoking habit on the urinary t,t-MA/SPMA ratio.

The identification of reliable biomarkers is critical for the assessment of occupational exposure of benzene: S-phenylmercapturic acid (SPMA) and trans,trans-muconic acid (t,t-MA) are the most currently used. t,t-MA is an open-ring metabolite, but it is also a metabolite of the food preservative sorbic acid, while SPMA is formed by conjugation with glutathione, and several studies suggested that the genetic polymorphism of glutathione S-transferases modulates its production. This study compared the ability of these metabolites to assess the benzene exposure in a big group of petrochemical workers. Furthermore, investigated how genetic polymorphism of glutathione S-transferase theta 1 (GSTT1), glutathione S-transferase mu 1 (GSTM1), glutathione S-transferase pi 1 (GSTP1) and smoking habits, may influence their excretion. Results showed that occupational exposure to benzene was negligible compared to that from smoking and confirmed the modulating effect of the genetic polymorphism of GSTT1 on the urinary excretion of SPMA, but not of t, t-MA, even at very low levels of benzene exposure. The same effect was found for GSTM1, but only for smokers. The t,t-MA/SPMA ratio was not a constant value and resulted to be higher than the corresponding Biological Exposure Index (BEI) ratio, which is currently equal to 20. Higher values of metabolite have been associated with the GSTT1 or GSTM1 null genotype and these are responsible for increase health risk. We suggest that this ratio could be used as a marker of individual susceptibility for subjects with benzene exposure.

Cytogenetic biomonitoring on a group of petroleum refinery workers.

Workers employed in petroleum refineries are exposed to a wide range of toxic compounds (benzene, polycyclic aromatic hydrocarbons, heavy metals, etc.) with known mutagenic and carcinogenic potential. In this study, we investigated by using the cytokinesis block micronucleus (CBMN) assay on human peripheral blood lymphocytes (PBL) whether general occupational exposure in petroleum refineries resulted in early biological effects, which would be indicative of adverse health effects in the long term. In this study, out of more 500 workers enrolled in the study, 79 male subjects (46 nonsmokers and 33 smokers), employed in two different Italian petroleum refineries, and a total of 50 male control subjects (34 nonsmokers and 16 smokers) were selected by using very strict selection criteria. The comparison of chromosome damage in PBL between exposed and control populations pointed out a significant increase of micronuclei in the exposed group, correlated with the length of employment. Results confirm that smoking is the principal confounding factor for the responses. In conclusion, our results are indicative of a potential genotoxic risk related to the complex occupational exposure in petroleum refineries, despite the measures adopted in the plants, and corroborate the need to increase safety measures to avoid exposure to chemical agents.

[Semi-permanent make up and tattooing equipment: safety and health issues]. / Apparecchiature per trucco semipermanente e tatuaggi: Requisiti per la salute e la sicurezza.

The UK's Health and Safety Executive (HSE), at Machinery Directive Administrative Cooperation meeting, brought a problem to the attention of semi-permanent make up equipment sold in the European market, that may pose a health risk due to microbiological contamination. Considering that body adornment techniques are becoming increasingly popular, a study was carried out to evaluate the technical characteristics of such equipment and to collect information on the National and European regulatory situation. In most member states of the European Community, tattooing and semi-permanent make up activities are not regulated by specific laws. In Italy, the "Guidelines for safe tattooing and piercing practices" were laid down by the Ministry of Health in 1998 (Notes N. 2.8/156 of 5 February 1998 and n.2.8/633 of 16 July 1998). This paper discusses the need to implement the above guidelines and to harmonize European legislation on the subject.

[Prevention of transmissible spongiform encephalopathy and methods for prion inactivation]. / Prevenzione delle encefalopatie spongiformi trasmissibili e metodi di inattivazione dei prioni.

BACKGROUND: Transmissible Spongiform Encephalopathies (TSEs) are infectious, progressive, lethal neurodegenerative diseases which affect both human and other mammalian species. The knowledge on the agent responsible for the infection and its pathogenetic mechanism is still limited. Specific diagnostic tests are currently not available; diagnosis is based on clinical symptoms and confirmed by a post-mortem examination which can reveal the typical brain lesions. There is some evidence on the relationship between the agent responsible for Bovine Spongiform Encephalopathy (BSE) and the new human variant of the Creutzfeldt-Jakob disease (nvCJD). The TSEs agents have some peculiarities: they overcome the interspecies barriers and are resistant to the normal disinfections and sterilization procedures. OBJECTIVES: On the basis of current scientific evidence, the aim of this article was: to make an excursus on the efficacy of inactivation methods and to give an overview of what has been issued by International and Italian Regulatory Agencies for the prevention of such diseases in hospital and occupational environments. METHODS: We examined current scientific literature on inactivation of TSEs by physical or chemical methods or combinations thereof. We also reviewed the most relevant guidelines on exposure risk, containment and occupational exposure to TSEs agents. RESULTS AND CONCLUSIONS: Due to the peculiarity of the prion protein and its transmission, it is very important to have effective methods to inactivate the TSE agents and to prevent them spreading. At present, no certain data are available on TSE development in occupational environments, while sources of exposure risk are known for several occupational categories, such as health-care personnel, pathologists, technicians in diagnostic and research laboratories, farmers, veterinary surgeons, slaughter house operators. For these workplaces, after an accurate risk assessment, it is necessary to implement certain precautionary measures, based on containment procedures and on the adoption of specific inactivation protocols.