Acinetobacter baumannii from grass: novel but non-resistant clones

Acinetobacter baumannii is one the most worrisome nosocomial pathogens, which has long been considered almost mainly as a hospital-associated bacterium. There have been some studies about animal and environmental isolates over the last decade. However, little effort has been made to determine if this pathogen dwells in the grass. Here, we aim to determine the evolutionary relationships and antibiotic resistance of clones of A. baumannii sampled from grass to the major human international clones and animal clones. Two hundred and forty genomes were considered in total from four different sources for this study. Our core and accessory genomic epidemiology analyses showed that grass isolates cluster in seven groups well differentiated from one another and from the major human and animal isolates. Furthermore, we found new sequence types under both multilocus sequence typing schemes: two under the Pasteur scheme and seven for the Oxford scheme. The grass isolates contained fewer antibiotic-resistance genes and were not resistant to the antibiotics tested. Our results demonstrate that these novel clones appear to have limited antibiotic resistance potential. Given our findings, we propose that genomic epidemiology and surveillance of A. baumannii should go beyond the hospital settings and consider the environment in an explicit One Health approach

MALDI-TOF mass spectroscopy of yeasts and filamentous fungi for research and diagnostics in the agricultural value chain

Abstract Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS; MALDI biotyping) has become a standard tool for the accurate, rapid, and economical identification of pathogens in the clinical diagnostics laboratory. The method is continuously being improved, and new applications for distinguishing strains, identifying metabolites or functional characteristics (e.g., antibiotic resistance), and detecting microbes directly in patient samples have been developed. Adopting these methods in other disciplines than clinical diagnostics, for example, in agriculture, food safety and quality testing, or ecology, will open up new opportunities for diagnostics and research. This review focuses on MALDI-TOF MS approaches for the identification of yeasts and filamentous fungi. In contrast to bacterial diagnostics, MALDI biotyping of fungi is more challenging and less established. We thus start by discussing the role of MALDI-TOF MS as a tool for species identification; in particular with respect to DNA-based identification methods. The review then highlights the value of custom-made reference spectra for MALDI biotyping and points out recent advancements of MALDI-TOF MS, mainly from the field of clinical diagnostics that may be adopted and used for fungal diagnostic challenges. The overview ends with a summary of MALDI-TOF MS studies of yeasts and filamentous fungi of agricultural relevance. Graphical abstract MALDI biotyping is the method of choice for the repeated, rapid identification of a defined number of species

Chasing the structures of small molecules in arbuscular mycorrhizal signaling

The arbuscular mycorrhiza (AM) is a symbiosis between most terrestrial plants and fungi of the ancient phylum Glomeromycota. AM improves the uptake of water and mineral nutrients, such as phosphorus (P) and nitrogen (N), of the host plant in exchange for photosynthetically fixed carbon. Successful colonization and a functional interaction between host plant and mycobiont are based upon exchange of signaling molecules at different stages of symbiosis development. Strigolactones, a novel class of plant hormones, are secreted by plant roots stimulating presymbiotic growth of AM fungi. Fungi release soluble signaling molecules, the enigmatic ‘Myc factors’, that activate early symbiotic root responses. Lysophosphatidylcholine is a lipophilic intraradical mycorrhizal signal triggering plant phosphate transporter gene expression late in AM development through a P-controlled transcriptional mechanism. This enables uptake of orthophosphate released from the AM fungus

Geobacillus and Bacillus Spore Inactivation by Low Energy Electron Beam Technology: Resistance and Influencing Factors

Low energy electron beam (LEEB) treatment is an emerging non-thermal technology that performs surface decontamination with a minimal influence on food quality. Bacterial spore resistance toward LEEB treatment and its influencing factors were investigated in this study. Spores from Geobacillus and Bacillus species were treated with a labscale LEEB at energy levels of 80 and 200 keV. The spore resistances were expressed as D-values (the radiation dose required for one log10 reduction at a given energy level) calculated from the linear regression of log10 reduction against absorbed dose of the sample. The results revealed that the spore inactivation efficiency by LEEB is comparable to that of other ionizing radiations and that the inactivation curves are mostly log10-linear at the investigated dose range (3.8 – 8.2 kGy at 80 keV; 6.0 – 9.8 kGy at 200 keV). The D-values obtained from the wildtype strains varied from 2.2 – 3.0 kGy at 80 keV, and from 2.2 – 3.1 kGy at 200 keV. Bacillus subtilis mutant spores lacking a/b-type small, acid-soluble spore proteins showed decreased D-values (1.3 kGy at 80 and 200 keV), indicating that spore DNA is one of the targets for LEEB spore inactivation. The results revealed that bacterial species, sporulation conditions and the treatment dose influence the spore LEEB inactivation. This finding indicates that for the application of this emerging technology, special attention should be paid to the choice of biological indicator, physiological state of the indicator and the processing settings. High spore inactivation efficiency supports the application of LEEB for the purpose of food surface decontamination. With its environmental, logistical, and economic advantages, LEEB can be a relevant technology for surface decontamination to deliver safe, minimally processed and additive-free food products

Tracing Antibiotic Resistance Genes along the Irrigation Water Chain to Chive: Does Tap or Surface Water Make a Difference?

Irrigation water is well known as potential source of pathogens in fresh produce. However, its role in transferring antibiotic resistance determinants is less well investigated. Therefore, we analyzed the contribution of surface and tap water to the resistome of overhead-irrigated chive plants. Field-grown chive was irrigated with either surface water (R-system) or tap water (D-system), from planting to harvest. Water along the two irrigation chains as well as the respective plants were repeatedly sampled and screened for 264 antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs), using high-capacity qPCR. Differentially abundant (DA) ARGs were determined by comparing the two systems. On R-chive, β-lactam ARGs, multidrug-resistance (MDR) determinants, and MGEs were most abundant, while D-chive featured DA ARGs from the vancomycin class. Diversity and number of DA ARGs was the highest on young chives, strongly diminished at harvest, and increased again at the end of shelf life. Most ARGs highly enriched on R- compared to D-chive were also enriched in R- compared to D-sprinkler water, indicating that water played a major role in ARG enrichment. Of note, blaKPC was detected at high levels in surface water and chive. We conclude that water quality significantly affects the resistome of the irrigated produce

Inactivation of mould spores in a model system and on raisins by low-energy electron beam

Low-energy electron beam was investigated as an intervention strategy for the reduction of spores of food-spoiling fungi. Inactivation kinetics of five strains from the genera Aspergillus, Byssochlamys, Eurotium and Penicillium were determined. A two-dimensional membrane was used as a model system to exclude matrix effects. Raisins were incorporated into the study as an example of a three-dimensional food surface structure that is regularly being affected by spoilage through moulds. Complete inactivation could be attained on the model system after a maximum dose of 6 kGy, whereas between 1.04 and 1.71 log reductions were achieved on raisins. This novel application of low-energy electron beam was shown to reduce mould spores even on challenging food surfaces, paving the way for implementation in food industry

Transferable Extended-Spectrum β-Lactamase (ESBL) Plasmids in Enterobacteriaceae from Irrigation Water

Extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae are classified as serious threats to human health by the U.S. Centers for Disease Control and Prevention. Water used for irrigation of fresh produce can transmit such resistant bacteria directly to edible plant parts. We screened ESBL-producing Escherichia coli, Enterobacter cloacae, and Citrobacter freundii isolated from irrigation water for their potential to transmit resistance to antibiotic-susceptible E. coli. All strains were genome-sequenced and tested in vitro for transmission of resistance to third-generation cephalosporins on solid agar as well as in liquid culture. Of the 19 screened isolates, five ESBL-producing E. coli were able to transfer resistance with different efficiency to susceptible recipient E. coli. Transconjugant strains were sequenced for detection of transferred antibiotic resistance genes (ARGs) and compared to the known ARG pattern of their respective donors. Additionally, phenotypic resistance patterns were obtained for both transconjugant and corresponding donor strains, confirming ESBL-producing phenotypes of all obtained transconjugants

Direct identification of Monilinia brown rot fungi on infected fruits by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry

Background: Brown rot of stone and pome fruit is a serious fungal disease that is mainly caused by four species in the genus Monilinia. Of these four species, Monilinia fructicola is the most devastating pathogen and of particular concern because it undergoes sexual recombination and has recently been introduced to Europe. So far, Monilinia diagnosis required a multiplex PCR analysis and gel electrophoresis. In contrast, intact-protein biotyping by mass spectrometry is considerably faster and cheaper. However, it usually requires an in vitro cultivation step prior to the MALDI analysis. It was thus attempted to establish a method for the identification of Monilinia species by MALDI biotyping with fungal material derived directly from infected fruits; without an in vitro cultivation step. Results: To simplify and render MALDI biotyping of fungi more reliable, an improved protocol for the preparation of crude protein extracts and for collecting MALDI-TOF MS data for biotyping was developed. We generated reference spectra for all four Monilinia brown rot fungi and were able to reliably identify Monilinia species based on fungal material that was collected directly from infected fruits. This method allowed the correct, fast and economic identification of M. fructicola and M. laxa, while M. fructigena and M. polystroma could not be distinguished reliably. Conclusions: MALDI biotyping may be used as an economical tool for the routine diagnosis of Monilinia brown rot fungi on infected fruits