Implementation of molecular techniques in the diagnosis of legionnaires’ disease and in the investigation of legionella outbreaks

Background. Legionnaires’ Disease (LD) is a mild to severe, potentially lethal, respiratory syndrome caused by members of the Legionella genus, in particular L. pneumophila serogroup (sg) 1 alone causes about 95% of culture confirmed cases. The infection is usually acquired by inhalation of aerosols originating from contaminated fresh water sources, consequently typing of both clinical and environmental isolates is crucial to rapidly identify the possible source and prevent further cases. Legionellae are difficult to isolate by culture, moreover as respiratory samples are not available for up to 65% patients, alternative techniques are needed to diagnose LD and maximise the amount of typing data that can be obtained to aid investigations. Urinary antigen detection and serology provide very limited information regarding the infecting strain, while the advent of PCR and Sanger sequencing has allowed reliable diagnostic and typing methods to be introduced. Objectives. The aim of this study was to improve existing diagnostic and typing molecular assays, and to develop new ones to further standardise diagnostic and typing procedures across members of the European Society for Clinical Microbiology and Infectious Diseases (ESCMID) Study Group for Legionella Infections (ESGLI). Utility of the assays was assessed in both routine and outbreak scenarios. Methods. A wide range of both in silico and in vitro experiments were used to design and validate specific oligonucleotides to improve detection and typing of L. pneumophila. Genomic DNA was manually extracted and prepared for Whole Genome Sequencing (WGS) using Illumina platforms. A bioinformatic approach was used to design a WGS based typing scheme and decipher the evolution of L. pneumophila sg1 Sequence Type (ST) 47, a major disease-causing strain. Results. A real-time PCR detecting L. pneumophila and sg1 specific targets was validated with international colleagues and made available to ESGLI members. Sequence based typing was improved and expanded, and specific typing guidelines produced. A 50 gene core-genome MLST was identified as the best approach to improve the current typing method. ST47 was shown to be a ‘chimera’ between ST109 and ST62, and a specific real-time PCR was designed to detect this strain. Conclusions. The results of this study allowed researchers to obtain faster and more accurate diagnosis of LD, and L. pneumophila typing data from both isolates and primary samples. A metagenomics approach is presently under evaluation to obtain typing results by WGS directly from clinical and environmental samples

Low genomic diversity of Legionella pneumophila within clinical specimens.

OBJECTIVES: Legionella pneumophila is the leading cause of Legionnaires' disease, a severe form of pneumonia acquired from environmental sources. Investigations of both sporadic cases and outbreaks rely mostly on analysis of a single to a few colony pick(s) isolated from each patient. However, because of the lack of data describing diversity within single patients, the optimal number of picks is unknown. Here, we investigated diversity within individual patients using sequence-based typing (SBT) and whole-genome sequencing (WGS). METHODS: Ten isolates of L. pneumophila were obtained from each of ten epidemiologically unrelated patients. SBT and WGS were undertaken, and single-nucleotide polymorphisms (SNPs) were identified between isolates from the same patient. RESULTS: The same sequence type (ST) was obtained for each set of ten isolates. Using genomic analysis, zero SNPs were identified between isolates from seven patients, a maximum of one SNP was found between isolates from two patients, and a maximum of two SNPs was found amongst isolates from one patient. Assuming that the full within-host diversity has been captured with ten isolates, statistical analyses showed that, on average, analysis of one isolate would yield a 70% chance of capturing all observed genotypes, and seven isolates would yield a 90% chance. CONCLUSIONS: SBT and WGS analyses of multiple colony picks obtained from ten patients showed no, or very low, within-host genomic diversity in L. pneumophila, suggesting that analysis of one colony pick per patient will often be sufficient to obtain reliable typing data to aid investigation of cases of Legionnaires' disease

MOLECULAR EPIDEMIOLOGY OF STENOTROPHOMONAS MALTOPHILIA (SM) IN THE CYSTIC FIBROSIS (CF) CENTRE OF GENOVA

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Sturgeon meat and caviar quality from different cultured species

Sturgeon raw eggs, caviar and meat obtained from different species reared in an Italian production plant were evaluated for their chemical composition, in order to improve their appreciation on the market and to detect any eventual distinctness related to the species. Mainly, fatty acid (FA) profile of eggs and caviar, determined by Gas\u2010Chromatography coupled to Flame Ionization Detection, showed variability in the interspecific comparison, highlighted by chemometric methods (Linear Discriminant Analysis). Generally, all samples showed a prevalence of unsaturated fatty acids with respect to saturated ones, reaching a content of polyunsaturated fatty acids (PUFA) between the 40% and the 50% of total FA. A remarkable presence of n3 series PUFA was detected in all samples and a selective deposition of many FA into eggs\u2019 cellular membranes, yolk lipid and body fat reserves, imputable to the different biological role of single FA during sturgeon reproduction, was evidenced. Chemical composition of sturgeon flesh samples evidenced a high\u2010protein and medium\u2010fat content, characterized by a FA profile of high nutritional value. Moreover, color parameters (redness, yellowness, brightness, Chroma) were measured on sturgeon fillets, showing many species\u2010specific characteristics of sturgeon meat

Volatile Organic Compounds Profile in White Sturgeon (Acipenser transmontanus) Caviar at Different Stages of Ripening by Multiple Headspace Solid Phase Microextraction

Caviar is considered a delicacy by luxury product consumers, but few data are available about its flavour chemistry to date. In this study, a multiple headspace-solid phase microextraction (MHS-SPME) followed by gas chromatography and mass spectrometry (GC-MS) approach was developed and employed to identify and quantitatively estimate key volatile organic compounds (VOCs) representative in white sturgeon (A. transmontanus) caviar at five different stages of ripening: raw eggs (t0), after 60 days (t1), 120 days (t2), 180 days (t3), and 240 days (t4) of ripening. The method showed the ability to detect and estimate the quantity of 25 flavour compounds, without any severe alteration of the matrix before the analysis and in a short time. The VOCs detected as representative in caviar samples were primarily aldehydes and alcohols, already well known as responsible of fresh fish and seafood flavours, and mainly deriving from lipid peroxidation processes and microbial activity against lipids and amino acids. We found a significant (p < 0.01) increase in the amount of total aldehydes within t0 (29.64 ng/g) and t4 (121.96 ng/g); moreover, an interesting, great arise of 3-hydroxy-2-butanone at the final stage of storage (48.17 ng/g) was recorded. Alcohols were not detected in raw eggs (t0) and then a decrease from t1 (17.77 ng/g) to t4 (10.18 ng/g) was recorded in their amount, with no statistical significance

The relative absorption of fatty acids in brown trout (Salmo trutta) fed a commercial extruded pellet coated with different lipid sources

The objective of the present study was to investigate the fatty acid absorption capabilities of brown trout (Salmo trut- ta) fed commercial extruded diets. Five commercial extruded pellets, different only in the lipid sources used for fat coat- ing, were tested on juvenile brown trout for 45 days. The trout were reared in fresh water at 14.6 ± 0.4° C and 7.7 ± 0.3 mg/l, temperature and dissolved oxygen, respectively. The tested lipid sources were fish oil, canola oil, oleine oil, swine fat and poultry fat. After the adaptation period faeces were collected by gently stripping from anaesthetized fish. Fatty acid analysis was performed on experimental diets and on collected faeces to evaluate the relative absorption capabilities of the trout digestive system with respect to each detected fatty acid. The use of the relative absorption efficiency (rAE) was opted to evaluate the intrinsic capability of each fatty acid to be absorbed. Brown trout showed a specific preferential order of absorption of the fatty acids, preferring shorter over longer chain fatty acids and prefer- ring the more unsaturated to the more saturated fatty acids. The fatty acid that showed the best relative absorbability was the C18:4n-3 (rAE = 5.14 ± 0.72), which has a fairly short carbon chain, but at the same time a high unsatura- tion level, followed by the C18:3n-3 (rAE = 3.38 ± 0.30). The fatty acid that showed the worst relative absorbability (rAE = 0.21 ± 0.02) was C24:1n-9