Transcriptional Profiling of Three Pseudomonas syringae pv. actinidiae Biovars Reveals Different Responses to Apoplast-Like Conditions Related to Strain Virulence on the Host.

Pseudomonas syringae pv. actinidiae is a phytopathogen that causes devastating bacterial canker in kiwifruit. Among five biovars defined by genetic, biochemical, and virulence traits, P. syringae pv. actinidiae biovar 3 (Psa3) is the most aggressive and is responsible for the most recent reported outbreaks; however, the molecular basis of its heightened virulence is unclear. Therefore, we designed the first P. syringae multistrain whole-genome microarray, encompassing biovars Psa1, Psa2, and Psa3 and the well-established model P. syringae pv. tomato, and analyzed early bacterial responses to an apoplast-like minimal medium. Transcriptomic profiling revealed i) the strong activation in Psa3 of all hypersensitive reaction and pathogenicity (hrp) and hrp conserved (hrc) cluster genes, encoding components of the type III secretion system required for bacterial pathogenicity and involved in responses to environmental signals; ii) potential repression of the hrp/hrc cluster in Psa2; and iii) activation of flagellum-dependent cell motility and chemotaxis genes in Psa1. The detailed investigation of three gene families encoding upstream regulatory proteins (histidine kinases, their cognate response regulators, and proteins with diguanylate cyclase or phosphodiesterase domains) indicated that cyclic di-GMP may be a key regulator of virulence in P. syringae pv. actinidiae biovars. The gene expression data were supported by the quantification of biofilm formation. Our findings suggest that diverse early responses to the host apoplast, even among bacteria belonging to the same pathovar, can lead to different virulence strategies and may explain the differing outcomes of infections. Based on our detailed structural analysis of hrp operons, we also propose a revision of hrp cluster organization and operon regulation in P. syringae.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The role of drug efflux pumps in Malassezia pachydermatis and Malassezia furfur defence against azoles.

This study aims to evaluate the effect of efflux pump modulators (EPMs) on the minimal inhibitory concentration (MIC) of fluconazole (FLZ) and voriconazole (VOR) in Malassezia furfur and Malassezia pachydermatis. The in vitro efficacy of azoles, in combination with EPMs (ie haloperidol-HAL, promethazine-PTZ and cyclosporine A-CYS), against 21 M. furfur from bloodstream infection patients and 14 M. pachydermatis from the skin of dogs with dermatitis, was assessed using a broth microdilution chequerboard analysis. Data were analysed using the model-fractional inhibitory concentration index (FICI) method. The MIC of FLZ and VOR of Malassezia spp. decreased in the presence of sub-inhibitory concentrations of HAL and/or PTZ. The synergic effect was observed only in strains with FLZ MIC≥128 µg/mL for M. furfur, FLZ MIC≥64 µg/mL for M. pachydermatis and VOR MIC≥4 µg/mL in both Malassezia spp. These results suggest that the drug efflux pumps are involved as defence mechanisms to azole drugs in Malassezia yeast. The synergism might be related to an increased expression of efflux pump genes, eventually resulting in azole resistance phenomena. Finally, the above FLZ and VOR MIC values might be considered the cut-off to discriminate susceptible and resistant strains.

Azole susceptibility of Malassezia pachydermatis and Malassezia furfur and tentative epidemiological cut-off values.

This study aims to determine the minimal inhibitory concentration (MIC) distribution and the epidemiological cut-off values (ECVs) of Malassezia pachydermatis and Malassezia furfur isolates for fluconazole (FLZ), itraconazole (ITZ), posaconazole (POS), and voriconazole (VOR). A total of 62 M. pachydermatis strains from dogs with dermatitis and 78 M. furfur strains from humans with bloodstream infections (BSI) were tested by a modified broth microdilution Clinical and Laboratory Standards Institute (CLSI) method. ITZ and POS displayed lower MICs than VOR and FLZ, regardless of the Malassezia species. The MIC data for azoles of M. pachydermatis were four two-fold dilutions lower than those of M. furfur. Based on the ECVs, about 94% of Malassezia strains might be categorized within susceptible population for all azoles, except for FLZ, and azole cross-resistance was detected in association with FLZ in M. pachydermatis but not in M. furfur.The study proposes, for the first time, tentative azole ECVs for M. pachydermatis and M. furfur for monitoring the emergence of isolates with decreased susceptibilities and shows that the azole MIC distribution varied according to the Malassezia species tested, thus suggesting the usefulness of determining the susceptibility profile for effective treatment of each species.

Cryptococcus neoformans in the respiratory tract of squirrels, Callosciurus finlaysonii (Rodentia, Sciuridae).

Cryptococcosis is a fungal disease acquired from the environment, for which animals may serve as sentinels for human exposure. The occurrence of Cryptococcus spp. in the respiratory tract of 125 squirrels, Callosciurus finlaysonii, trapped in Southern Italy, was assessed. Upon examination of nasal swabs and lung tissue from each individual, a total of 13 (10.4%) animals scored positive for yeasts, 7 for Cryptococcus neoformans (C.n.) (5.6%) and 6 for other yeasts (4.8%). C.n. was isolated from the nostrils and lungs, with a high population size in nostrils. Two C.n. molecular types, VNI and VNIV, were identified, with C.n. var. grubii VNI the most prevalent. Phylogenetic analyses of ITS+ and URA5 sequences revealed that C.n. isolates were genetically similar to isolates from a range of geographical areas and hosts. Results suggest that C.n. can colonize or infect the respiratory tract of C. finlaysonii. The high occurrence and level of colonization of nasal cavities might be an indicator of environmental exposure to high levels of airborne microorganism. The close phylogenetic relationship of C.n. strains from squirrels with those from human and other animal hosts suggests a potential role for these animals as "sentinels" for human exposure.

Plant Signals Anticipate the Induction of the Type III Secretion System in Pseudomonas syringae pv. actinidiae, Facilitating Efficient Temperature-Dependent Effector Translocation.

Disease resistance in plants depends on a molecular dialogue with microbes that involves many known chemical effectors, but the time course of the interaction and the influence of the environment are largely unknown. The outcome of host-pathogen interactions is thought to reflect the offensive and defensive capabilities of both players. When plants interact with Pseudomonas syringae, several well-characterized virulence factors contribute to early bacterial pathogenicity, including the type III secretion system (T3SS), which must be activated by signals from the plant and environment to allow the secretion of virulence effectors. The manner in which these signals regulate T3SS activity is still unclear. Here, we strengthen the paradigm of the plant-pathogen molecular dialogue by addressing overlooked details concerning the timing of interactions, specifically the role of plant signals and temperature on the regulation of bacterial virulence during the first few hours of the interaction. Whole-genome expression profiling after 1 h revealed that the perception of plant signals from kiwifruit or tomato extracts anticipated T3SS expression in P. syringae pv. actinidiae compared to apoplast-like conditions, facilitating more efficient effector transport in planta, as revealed by the induction of a temperature-dependent hypersensitive response in the nonhost plant Arabidopsis thaliana Columbia-0 (Col-0). Our results show that in the arms race between plants and bacteria, the temperature-dependent timing of bacterial virulence versus the induction of plant defenses is probably one of the fundamental parameters governing the outcome of the interaction. IMPORTANCE Plant diseases-their occurrence and severity-result from the impact of three factors: the host, the pathogen, and the environmental conditions, interconnected in the disease triangle. Time was further included as a fourth factor accounting for plant disease, leading to a more realistic three-dimensional disease pyramid to represent the evolution of disease over time. However, this representation still considers time only as a parameter determining when and to what extent a disease will occur, at a scale from days to months. Here, we show that time is a factor regulating the arms race between plants and pathogens, at a scale from minutes to hours, and strictly depends on environmental factors. Thus, besides the arms possessed by pathogens and plants per se, the opportunity and the timing of arms mobilization make the difference in determining the outcome of an interaction and thus the occurrence of plant disease.

Application of Chemical Genomics to Plant-Bacteria Communication: A High-Throughput System to Identify Novel Molecules Modulating the Induction of Bacterial Virulence Genes by Plant Signals.

The life cycle of bacterial phytopathogens consists of a benign epiphytic phase, during which the bacteria grow in the soil or on the plant surface, and a virulent endophytic phase involving the penetration of host defenses and the colonization of plant tissues. Innovative strategies are urgently required to integrate copper treatments that control the epiphytic phase with complementary tools that control the virulent endophytic phase, thus reducing the quantity of chemicals applied to economically and ecologically acceptable levels. Such strategies include targeted treatments that weaken bacterial pathogens, particularly those inhibiting early infection steps rather than tackling established infections. This chapter describes a reporter gene-based chemical genomic high-throughput screen for the induction of bacterial virulence by plant molecules. Specifically, we describe a chemical genomic screening method to identify agonist and antagonist molecules for the induction of targeted bacterial virulence genes by plant extracts, focusing on the experimental controls required to avoid false positives and thus ensuring the results are reliable and reproducible.

Laboratory evaluation of a native strain of Beauveria bassiana for controlling Dermanyssus gallinae (De Geer, 1778) (Acari: Dermanyssidae).

The poultry red mite, Dermanyssus gallinae (De Geer, 1778) (Acari: Dermanyssidae) is one of the most economically important ectoparasites of laying hens worldwide. Chemical control of this mite may result in environmental and food contamination, as well as the development of drug resistance. High virulence of Beauveria bassiana sensu lato strains isolated from naturally infected hosts or from their environment has been demonstrated toward many arthropod species, including ticks. However, a limited number of studies have assessed the use of B. bassiana for the control of D. gallinae s.l. and none of them have employed native strains. This study reports the pathogenicity of a native strain of B. bassiana (CD1123) against nymphs and adults of D. gallinae. Batches of nymph and adult mites (i.e., n=720 for each stage) for treated groups (TGs) were placed on paper soaked with a 0.1% tween 80 suspension of B. bassiana (CIS, 10(5), 10(7) and 10(9) conidia/ml), whilst 240 untreated control mites for each stage (CG) were exposed only to 0.1% tween 80. The mites in TG showed a higher mortality at all stages (p<0.01) when compared to CG, depending on the time of exposure and the conidial concentration. A 100% mortality rate was recorded using a CIS of 10(9) conidia/ml 12 days post infection (DPI) in adults and 14 DPI in nymphs. B. bassiana suspension containing 10(9) conidia/ml was highly virulent towards nymph and adult stages of D. gallinae, therefore representing a possible promising natural product to be used in alternative or in combination to other acaricidal compounds currently used for controlling the red mite.