Genome-based typing reveals rare events of patient contamination with Pseudomonas aeruginosa from other patients and sink traps in a medical intensive care unit.

AIM: We used genome-based typing data with the aim of identifying the routes of acquisition of Pseudomonas aeruginosa by patients hospitalized in a medical intensive care unit (MICU) over a long period in a non-epidemic context. METHODS: This monocentric prospective study took place over 10 months in 2019 in a 15-bed MICU that applies standard precautions of hygiene. Lockable sink traps installed at all water points of use were bleach disinfected twice a week. We sampled all sink traps weekly to collect 404 P. aeruginosa environmental isolates and collected all P. aeruginosa isolates (N = 115) colonizing or infecting patients (N = 65). All isolates had their phenotypic resistance profile determined and their genome sequenced, from which we identified resistance determinants and assessed the population structure of the collection at the nucleotide level to identify events of P. aeruginosa transmission. FINDINGS: All sink traps were positive for P. aeruginosa, each sink trap being colonized for several months by one or more clones. The combination of genomic and spatiotemporal data identified one potential event of P. aeruginosa transmission from a sink trap to a patient (1/65, 1.5%) and six events of patient cross-transmission, leading to the contamination of five patients (5/65, 7.7%). All transmitted isolates were fully susceptible to ß-lactams and aminoglycosides. CONCLUSIONS: Genome-based typing revealed the contamination of patients by P. aeruginosa originating from sink traps to be infrequent (1.5%) in an MICU with sink trap-bleaching measures, and that only 7.7% of the patients acquired P. aeruginosa originating from another patient.

Comparison of pulsed-field gel electrophoresis and whole-genome-sequencing-based typing confirms the accuracy of pulsed-field gel electrophoresis for the investigation of local Pseudomonas aeruginosa outbreaks.

AIM: To determine whether pulsed-field gel electrophoresis (PFGE) accurately recognizes isolates belonging to clusters defined by techniques based on whole-genome sequencing (WGS) using Pseudomonas aeruginosa as a model. METHODS: We selected 65 isolates of ST395 P. aeruginosa isolated in seven European hospitals between 1998 and 2012. Isolates were typed by PFGE and sequenced by WGS. A core genome multi-locus sequence typing (cgMLST) analysis based on 3831 genes was performed with a homemade pipeline. FINDINGS: PFGE identified eight pulsotypes and cgMLST differentiated nine clusters and nine singletons. Five cgMLST clusters and pulsotypes (31/65 isolates) coincided perfectly. Isolates without evident epidemiological links grouped by PFGE were separated by cgMLST (16/65 isolates) differentiating cities, suggesting that PFGE should be kept for the investigation of local outbreaks. Importantly, hypermutator isolates still shared the pulsotype with their parents (16/65 isolates), whereas they were not recognized by cgMLST. This shows that PFGE was less affected than WGS-based typing by the accelerated genetic drift that occurs in epidemic P. aeruginosa. CONCLUSIONS: although WGS-based typing has logically become the new reference standard, we show here that the PFGE can be used with confidence for the investigation of local outbreaks caused by P. aeruginosa.

Global emergence of the widespread Pseudomonas aeruginosa ST235 clone.

OBJECTIVES: Despite the non-clonal epidemic population structure of Pseudomonas aeruginosa, several multi-locus sequence types are distributed worldwide and are frequently associated with epidemics where multidrug resistance confounds treatment. ST235 is the most prevalent of these widespread clones. In this study we aimed to understand the origin of ST235 and the molecular basis for its success. METHODS: The genomes of 79 P. aeruginosa ST235 isolates collected worldwide over a 27-year period were examined. A phylogenetic network was built, using a Bayesian approach to find the Most Recent Common Ancestor, and we identified antibiotic resistance determinants and ST235-specific genes. RESULTS: Our data suggested that the ST235 sublineage emerged in Europe around 1984, coinciding with the introduction of fluoroquinolones as an antipseudomonal treatment. The ST235 sublineage seemingly spread from Europe via two independent clones. ST235 isolates then appeared to acquire resistance determinants to aminoglycosides, ß-lactams and carbapenems locally. Additionally, we found that all the ST235 genomes contained the exoU-encoded exotoxin and identified 22 ST235-specific genes clustering in blocks and implicated in transmembrane efflux, DNA processing and bacterial transformation. These unique combinations of genes may have contributed to the poor outcome associated with P. aeruginosa ST235 infections and increased the ability of this international clone to acquire mobile resistance elements. CONCLUSION: Our data suggest that P. aeruginosa ST235 (a) has become prevalent across the globe potentially due to the selective pressure of fluoroquinolones and (b) readily became resistant to aminoglycosides, ß-lactams and carbapenems through mutation and acquisition of resistance elements among local populations.

Common proteins located in pigeon, budgerigar, and hen droppings related to bird fancier's lung

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Microbiological consequences of indoor composting.

Recycling of organic waste appeals to more and more people. The aim of this study was to evaluate the microbiological contamination around organic waste bins at three distances over a 12-month period. Contamination near the customary trash of control households was evaluated at the beginning to ensure that there is no recruitment bias. Air samples using the MAS 100 impactor were carried out in 38 dwellings that do household waste composting and in 10 dwellings of controls. Collection of particles by CIP 10 rotating cup sampler and dust samples collected by electrostatic dust collector cloths were acquired in dwellings that do household waste composting. Samples were analyzed by culture and by real-time quantitative PCR. Information about dwelling characteristics and inhabitant practices was obtained by a standardized questionnaire. The genera most often isolated were Penicillium, Aspergillus, Cladosporium and Streptomyces. Near the organic waste bins, bioaerosol samples showed an increase of Acarus siro (P = 0.001). Sedimented dust analyses highlighted an increase of A. siro, Wallemia sebi, Aspergillus versicolor, and Cladosporium sphaerospermum concentrations after a 12-month survey compared to the beginning. Composting favors microorganism development over time, but does not seem to have an effect on the bioaerosol levels and the surface microbiota beyond 0.5 m from the waste bin.

Microbiological characterization of 3193 French dwellings of Elfe cohort children.

Although exposure to indoor microorganisms in early life has already been associated with respiratory illness or allergy protection, only a few studies have performed standardized samplings and specific microbial analysis. Moreover, most do not target the different groups of microorganisms involved in respiratory diseases (fungi, bacteria, dust mites). In our study, ten specific qPCR targets (6 fungal species, 1 family and 2 genera of bacteria, 1 house dust mite) were used to analyze the microorganism composition of electrostatic dust fall collector (EDC) from 3193 dwellings of the Elfe French cohort study. Multivariate analyses allowed us to show that the microbial composition of dwellings, assessed with simultaneous analysis of 10 microorganisms, can be characterized by four entities: three bacteria, house dust mite Dermatophagoïdes pteronyssinus, fungi Alternaria alternata, and five other molds. Some dwellings' intrinsic characteristics (occupational ratio, type of dwelling and presence of pets) clearly influence microorganism distribution, and six different profiles of dwellings, characterized by their composition in microorganisms, have been described across France. The use of these clusters seems promising in the evaluation of allergic risk. Allergic respiratory diseases will develop in the near future in some children of the Elfe cohort and will indicate to what extent our approach can be predictive of respiratory disease.

Protective proteins are differentially expressed in tomato genotypes differing for their tolerance to low-temperature storage.

When stored at low temperature, tomato fruits exhibit chilling injury symptoms, such as rubbery texture and irregular ripening. To identify proteins related to chilling tolerance, we compared two tomato near isogenic lines differing for their texture phenotype at harvest in a fruit-storage trial including two temperatures (4 and 20 degrees C) along several days of conservation. Fruit evolution was followed by assessing fruit color, ethylene emission and texture parameters. The most contrasted samples were submitted to proteomic analysis including two-dimensional electrophoresis and mass spectrometry of protein spots to identify the proteins, whose expression varied according to the genotype or the storage conditions. Unexpectedly, the most firm genotype at harvest was the most sensitive to cold storage. The other genotype exhibited a delay in fruit firmness loss leading to the texture differences observed after 20 days of 4 degrees C storage. The proteome analysis of these contrasted fruits identified 85 proteins whose quantities varied with temperature or genotype. As expected, cold storage decreased the expression of proteins related to maturation process, such as acidic invertase, possibly controlled post-translational regulation of polygalacturonase and up-regulated proteins related to freezing tolerance. However, the study point out proteins involved in the differential resistance to chilling conditions of the two lines. This includes specific isoforms among the large family of small heat shocked proteins, and a set of proteins involved in the defense against of the reticulum endoplasmic stress.

Analysis of plant phosphoproteins.

Chromatography supports to purify phosphorylated proteins (P-proteins) have become available recently, yet this has not been thoroughly investigated in the case of plant materials. In this study we used a commercial affinity matrix (Qiagen) and a test plant enzyme (phosphoenolpyruvate carboxylase PEPC). The malate test and gel blot experiments probed with a specific antibody (antiphosphorylated N-terminal domain) showed that the column efficiently binds P-PEPC from Sorghum with little or no contamination by non-P-PEPC. Similar results were obtained with the low-abundance PEPC of Arabidopsis leaves when a gel filtration step (Sephadex G-200) was performed prior to the chromatography. Three-dimensional mass spectrometry analysis of immunoprecipitated PEPC in Qiagen fractions confirmed this observation. Denaturing protein extraction by cold acetone/trichloroacetic acid of fixed material led to a complete, one-step separation of P-PEPC and non-P-PEPC. At a global scale, the column captured most of the (32)P-phosphate-labeled proteins in vivo (80%), the majority of which were subsequently found in the elution fraction (88%). This was also visualized by SDS-PAGE (1D and 2D gels) followed by Pro-Q diamond staining. Analysis of the P-protein fraction by 1D gels and liquid chromatography/tandem mass spectrometry allowed the identification of 250 proteins belonging to various functional categories. These results validate the method for in vitro/in vivo studies of native/denatured individual proteins/enzymes regulated by phosphorylation and for phosphorylome studies.