Hypersensitivity pneumonitis associated with inhalation of Lycoperdon spores (lycoperdonosis) in a 3-month-old English setter dog in Quebec.

A 3-month-old female English setter dog was presented to the Faculty of Veterinary Medicine of the Université de Montréal (Quebec) with acute respiratory distress. The dog had moderately increased C-reactive protein concentrations, and thoracic radiographs revealed a moderate, caudodorsal, nodular-to-miliary alveolo-interstitial pulmonary pattern that was worse in the perihilar region. Initial differential diagnoses included a fungal pneumonia (e.g., blastomycosis or histoplasmosis). Cytology of the bronchoalveolar lavage revealed several round, green structures ~2 µm in diameter, consistent with fungal spores. The dog was hospitalized, but within 24 h the respiratory condition deteriorated and euthanasia was elected. Post-mortem panfungal PCR and sequencing tests identified the spores as Lycoperdon sp. Retrospectively, the owners recalled that the dog had played in a wood pile with mushrooms and had sneezed in a cloud of spores, implying inhalation of Lycoperdon spores. This is the first report of a confirmed case of canine lycoperdonosis in eastern Canada (Quebec), and the radiographic features in this case differed slightly from previous reports. Diagnosis before bronchoalveolar lavage analysis was challenging, as spore inhalation was not initially reported. Although the disease is infrequently reported in dogs, this case report reminds veterinarians to consider lycoperdonosis as a differential diagnosis when addressing animals presented with acute dyspnea with similar radiographic lesions, and highlights the importance of history and cytology in diagnosing this condition. Key clinical message: Hypersensitivity pneumonitis secondary to inhalation of Lycoperdon spores must be included in differential diagnoses for a dog with acute onset of respiratory signs and a nodular-to-miliary interstitial pulmonary pattern coalescing in patchy perihilar alveolar pulmonary lesions, and should prompt clinicians to question owners regarding inhalation of mushroom spores.Although cytological examination of a bronchoalveolar lavage reveals the presence of fungal spores, panfungal PCR and sequencing tests are needed to pinpoint the species involved.

Pneumopathie d'hypersensibilité associée à l'inhalation de spores de Lycoperdon (lycoperdonose) chez un chien setter anglais de 3 mois au Québec. Une chienne setter anglais âgée de 3 mois a été présentée à la Faculté de médecine vétérinaire de l'Université de Montréal (Québec) avec une détresse respiratoire aiguë. Le chien présentait des concentrations de protéine C-réactive modérément augmentées et les radiographies thoraciques ont révélé un schéma pulmonaire alvéolo-interstitiel modéré, caudodorsal, nodulaire à miliaire, pire dans la région périhilaire. Les diagnostics différentiels initiaux incluaient une pneumonie fongique (par exemple, blastomycose ou histoplasmose). La cytologie du lavage broncho-alvéolaire a révélé plusieurs structures rondes et vertes d'environ 2 µm de diamètre, compatibles avec des spores fongiques. Le chien a été hospitalisé, mais en 24 heures, l'état respiratoire s'est détérioré et l'euthanasie a été décidée. Les tests panfongiques PCR et de séquençage post-mortem ont identifié les spores comme étant Lycoperdon sp. Rétrospectivement, les propriétaires ont mentionné que le chien avait joué dans un tas de bois avec des champignons et avait éternué dans un nuage de spores, ce qui implique une inhalation de spores de Lycoperdon. Il s'agit du premier rapport d'un cas confirmé de lycoperdonose canine dans l'est du Canada (Québec), et les caractéristiques radiographiques de ce cas différaient légèrement des rapports précédents. Le diagnostic avant l'analyse du lavage broncho-alvéolaire était difficile, car l'inhalation de spores n'avait pas été initialement signalée. Bien que la maladie soit rarement rapportée chez les chiens, ce rapport de cas rappelle aux vétérinaires de considérer la lycoperdonose comme un diagnostic différentiel lorsqu'ils traitent des animaux présentant une dyspnée aiguë avec des lésions radiographiques similaires, et souligne l'importance de l'anamnèse et de la cytologie dans le diagnostic de cette affection.Message clinique clé : La pneumopathie d'hypersensibilité secondaire à l'inhalation de spores de Lycoperdon doit être incluse dans les diagnostics différentiels chez un chien présentant un début aigu de signes respiratoires et un schéma pulmonaire interstitiel nodulaire à miliaire fusionnant dans des lésions pulmonaires alvéolaires périhilaires inégales, et devrait inciter les cliniciens à interroger les propriétaires concernant l'inhalation de spores de champignons.Bien que l'examen cytologique d'un lavage broncho-alvéolaire révèle la présence de spores fongiques, des tests panfongiques PCR et de séquençage sont nécessaires pour identifier les espèces impliquées.(Traduit par Dr Serge Messier).

Use of tetrasodium EDTA acid for the treatment of intraluminal obstruction of subcutaneous ureteral bypass devices.

OBJECTIVES: The aim of this study was to evaluate the efficacy and tolerability of a 4% tetrasodium EDTA (tEDTA) infusion protocol in the subcutaneous ureteral bypass (SUB) devices of cats with intraluminal obstruction at a veterinary teaching hospital between July 2017 and April 2020. METHODS: This was a retrospective controlled study. Cats with an obstructed SUB device underwent a 4% tEDTA infusion protocol. Obstruction of the device was diagnosed based on renal pelvic dilation, dilatation of the ureter, mineralized material within the device (cystostomy or nephrostomy catheters) seen on ultrasound, the absence of visible bubbles within the renal pelvis and/or urinary bladder following ultrasound-guided flushing of the device with saline. RESULTS: A total of 16 tEDTA infusion protocols were performed in 14 cats. The infusion protocol was considered successful in 11/16 SUB devices (68.8%). Six devices (n = 6/11; 54.5%) had recurrence of obstruction with a median time of 87 days. One or more episodes of self-limiting pollakiuria and/or hematuria following infusion was seen in eight patients (n = 8/14; 57.1%). CONCLUSIONS AND RELEVANCE: Infusions of 4% tEDTA successfully relieved intraluminal obstruction in patients with occluded SUB devices; however, the recurrence of obstruction was common. Additional studies evaluating case selection and optimal protocols are warranted.

A Novel Regulatory Cascade Involving BluR, YcgZ, and Lon Controls the Expression of Escherichia coli OmpF Porin.

In Escherichia coli, OmpF is an important outer membrane protein, which serves as a passive diffusion pore for small compounds including nutrients, antibiotics, and toxic compounds. OmpF expression responds to environmental changes such as temperature, osmolarity, nutrients availability, and toxic compounds via complex regulatory pathways involving transcriptional and post-transcriptional regulation. Our study identified a new regulatory cascade that controls the expression of OmpF porin. This pathway involves BluR, a transcriptional regulator repressing the expression of the ycgZ-ymgABC operon. We showed that BluR was responsible for the temperature-dependent regulation of the ycgZ-ymgABC operon. Furthermore, our results showed that independent expression of YcgZ led to a decreased activity of the ompF promoter, while YmgA, YmgB, and YmgC expression had no effect. We also determined that YcgZ accumulates in the absence of the Lon protease. Thus, mutation in bluR leads to de-repression of ycgZ-ymgABC transcription. With a second mutation in lon, YcgZ protein accumulates to reach levels that do not allow increased expression of OmpF under growth conditions that usually would, i.e., low temperature. With BluR responding to blue-light and temperature, this study sheds a new light on novel signals able to regulate OmpF porin.

The contribution of PmrAB to the virulence of a clinical isolate of Escherichia coli.

Previous data from our laboratory suggest a relationship between increased pmrAB expression and virulence in an Escherichia coli mouse infection model of pyelonephritis. Competitive infections with wild type and pmrAB mutants showed that disruption of pmrAB caused decreased persistence of E. coli within the mouse kidney. These results were confirmed with plasmid-mediated complementation of the pmrAB mutant. Additionally, increased expression of pmrAB from this complementing plasmid in a previously attenuated marA-rob-soxS triple mutant displayed increased bacterial persistence in the infection when compared with the triple mutant alone. These findings suggest a role for this two-component regulatory system in the virulence of E. coli in a murine pyelonephritis model.

Mutational analysis of the multiple-antibiotic resistance regulator MarR reveals a ligand binding pocket at the interface between the dimerization and DNA binding domains.

The Escherichia coli regulator MarR represses the multiple-antibiotic resistance operon marRAB and responds to phenolic compounds, including sodium salicylate, which inhibit its activity. Crystals obtained in the presence of a high concentration of salicylate indicated two possible salicylate sites, SAL-A and SAL-B. However, it was unclear whether these sites were physiologically significant or were simply a result of the crystallization conditions. A study carried out on MarR homologue MTH313 suggested the presence of a salicylate binding site buried at the interface between the dimerization and the DNA-binding domains. Interestingly, the authors of the study indicated a similar pocket conserved in the MarR structure. Since no mutagenesis analysis had been performed to test which amino acids were essential in salicylate binding, we examined the role of residues that could potentially interact with salicylate. We demonstrated that mutations in residues shown as interacting with salicylate at SAL-A and SAL-B in the MarR-salicylate structure had no effect on salicylate binding, indicating that these sites were not the physiological regulatory sites. However, some of these residues (P57, R86, M74, and R77) were important for DNA binding. Furthermore, mutations in residues R16, D26, and K44 significantly reduced binding to both salicylate and 2,4-dinitrophenol, while a mutation in residue H19 impaired the binding to 2,4-dinitrophenol only. These findings indicate, as for MTH313, the presence of a ligand binding pocket located between the dimerization and DNA binding domains.

Involvement of MarR and YedS in carbapenem resistance in a clinical isolate of Escherichia coli from China.

A carbapenem-resistant clinical isolate of Escherichia coli, which lacked OmpF and OmpC porins, carried a marR mutation and expressed a functional yedS, a normally nontranslated gene. MarR and YedS are described here as having effects on the ability of this strain to resist carbapenems. Additionally, expression of YedS was regulated by the small RNA MicF in a MarA-dependent way. These findings illustrate how broadly bacteria can mutate within a selective clinical setting, in this case, resistance to carbapenems, by altering three porin genes and one regulatory gene.

MarA, SoxS and Rob of Escherichia coli - Global regulators of multidrug resistance, virulence and stress response.

Bacteria have a great capacity for adjusting their metabolism in response to environmental changes by linking extracellular stimuli to the regulation of genes by transcription factors. By working in a co-operative manner, transcription factors provide a rapid response to external threats, allowing the bacteria to survive. This review will focus on transcription factors MarA, SoxS and Rob in Escherichia coli, three members of the AraC family of proteins. These homologous proteins exemplify the ability to respond to multiple threats such as oxidative stress, drugs and toxic compounds, acidic pH, and host antimicrobial peptides. MarA, SoxS and Rob recognize similar DNA sequences in the promoter region of more than 40 regulatory target genes. As their regulons overlap, a finely tuned adaptive response allows E. coli to survive in the presence of different assaults in a co-ordinated manner. These regulators are well conserved amongst Enterobacteriaceae and due to their broad involvement in bacterial adaptation in the host, have recently been explored as targets to develop new anti-virulence agents. The regulators are also being examined for their roles in novel technologies such as biofuel production.

A peptidoglycan fragment triggers ß-lactam resistance in Bacillus licheniformis.

To resist to ß-lactam antibiotics Eubacteria either constitutively synthesize a ß-lactamase or a low affinity penicillin-binding protein target, or induce its synthesis in response to the presence of antibiotic outside the cell. In Bacillus licheniformis and Staphylococcus aureus, a membrane-bound penicillin receptor (BlaR/MecR) detects the presence of ß-lactam and launches a cytoplasmic signal leading to the inactivation of BlaI/MecI repressor, and the synthesis of a ß-lactamase or a low affinity target. We identified a dipeptide, resulting from the peptidoglycan turnover and present in bacterial cytoplasm, which is able to directly bind to the BlaI/MecI repressor and to destabilize the BlaI/MecI-DNA complex. We propose a general model, in which the acylation of BlaR/MecR receptor and the cellular stress induced by the antibiotic, are both necessary to generate a cell wall-derived coactivator responsible for the expression of an inducible ß-lactam-resistance factor. The new model proposed confirms and emphasizes the role of peptidoglycan degradation fragments in bacterial cell regulation.

Backbone 1H, 13C, and 15N resonance assignments for lysozyme from bacteriophage lambda.

Lysozyme from lambda bacteriophage (lambda lysozyme) is an 18 kDa globular protein displaying some of the structural features common to all lysozymes; in particular, lambda lysozyme consists of two structural domains connected by a helix, and has its catalytic residues located at the interface between these two domains. An interesting feature of lambda lysozyme, when compared to the well-characterised hen egg-white lysozyme, is its lack of disulfide bridges; this makes lambda lysozyme an interesting system for studies of protein folding. A comparison of the folding properties of lambda lysozyme and hen lysozyme will provide important insights into the role that disulfide bonds play in the refolding pathway of the latter protein. Here we report the (1)H, (13)C and (15)N backbone resonance assignments for lambda lysozyme by heteronuclear multidimensional NMR spectroscopy. These assignments provide the starting point for detailed investigation of the refolding pathway using pulse-labelling hydrogen/deuterium exchange experiments monitored by NMR.

Combined inactivation of lon and ycgE decreases multidrug susceptibility by reducing the amount of OmpF porin in Escherichia coli.

Transposon inactivation of ycgE, a gene encoding a putative transcriptional regulator, led to decreased multidrug susceptibility in an Escherichia coli lon mutant. The multidrug susceptibility phenotype (e.g., to tetracycline and beta-lactam antibiotics) required the inactivation of both lon and ycgE. In this mutant, a decreased amount of OmpF porin contributes to the lowered drug susceptibility, with a greater effect at 26 degrees C than at 37 degrees C.

Activities of ceftobiprole and other cephalosporins against extracellular and intracellular (THP-1 macrophages and keratinocytes) forms of methicillin-susceptible and methicillin-resistant Staphylococcus aureus.

Staphylococcus aureus is an opportunistic intracellular organism. Although they poorly accumulate in eukaryotic cells, beta-lactams show activity against intracellular methicillin (methicillin)-susceptible S. aureus (MSSA) if the exposure times and the drug concentrations are sufficient. Intraphagocytic methicillin-resistant S. aureus (MRSA) strains are susceptible to penicillins and carbapenems because the acidic pH favors the acylation of PBP 2a by these beta-lactams through pH-induced conformational changes. The intracellular activity (THP-1 macrophages and keratinocytes) of ceftobiprole, which shows almost similar in vitro activities against MRSA and MSSA in broth, was examined against a panel of hospital-acquired and community-acquired MRSA strains (MICs, 0.5 to 2.0 mg/liter at pH 7.4 and 0.25 to 1.0 mg/liter at pH 5.5) and was compared with its activity against MSSA isolates. The key pharmacological descriptors {relative maximal efficacy (E(max)), relative potency (the concentration causing a reduction of the inoculum halfway between E(0) and E(max) [EC(50)]), and static concentration (C(s))} were measured. All strains showed sigmoidal dose-responses, with E(max) being about a 1 log(10) CFU decrease from the postphagocytosis inoculum, and EC(50) and C(s) being 0.2 to 0.3x and 0.6 to 0.9x the MIC, respectively. Ceftobiprole effectively competed with Bocillin FL (a fluorescent derivative of penicillin V) for binding to PBP 2a at both pH 5.5 and pH 7.4. In contrast, cephalexin, cefuroxime, cefoxitin, or ceftriaxone (i) were less potent in PBP 2a competitive binding assays, (ii) showed only partial restoration of the activity against MRSA in broth at acidic pH, and (iii) were collectively less effective against MRSA in THP-1 macrophages and were ineffective in keratinocytes. The improved activity of ceftobiprole toward intracellular MRSA compared with the activities of conventional cephalosporins can be explained, at least in part, by its greater ability to bind to PBP 2a not only at neutral but also at acidic pH.

Large ring 1,3-bridged 2-azetidinones: experimental and theoretical studies.

The relationship between angular strain and (re)activity of bicyclic 2-azetidinones is still an open question of major concern in the field of penicillin antibiotics. Our study deals with original 13-membered-ring 1,3-bridged 2-azetidinones related to the carbapenem family, and featuring a "planar amide" instead of the "twisted amide" typical of penam derivatives. The bicycles 11 and 12 were obtained from acetoxy-azetidinone 7, via the key-intermediate 10, by using the RCM (ring closing metathesis) strategy. Theoretical predictions and experimental results of hydrolysis showed that the large bicycle 12, endowed with high conformational flexibility, is more reactive than the bicycle 11, including a CC bond of E configuration, and the monocyclic 2-azetidinone precursor 10. The processing of 2-azetidinones 10-12 in the active site of serine enzymes has been computed by ab initio methods, considering three models. Due to geometrical parameters of the enzymic cavity (nucleophilic attack from the alpha-face), precursor 10 was predicted more active than 11 and 12 in the acylation step by Ser-OH. Indeed, bicycles 11 and 12 are modest inhibitors of PBP(2a), while 10 is a good to excellent inhibitor of PBP(2a) and R39 bacterial enzymes.

Conformational and thermodynamic changes of the repressor/DNA operator complex upon monomerization shed new light on regulation mechanisms of bacterial resistance against beta-lactam antibiotics.

In absence of beta-lactam antibiotics, BlaI and MecI homodimeric repressors negatively control the expression of genes involved in beta-lactam resistance in Bacillus licheniformis and in Staphylococcus aureus. Subsequently to beta-lactam presence, BlaI/MecI is inactivated by a single-point proteolysis that separates its N-terminal DNA-binding domain to its C-terminal domain responsible for its dimerization. Concomitantly to this proteolysis, the truncated repressor acquires a low affinity for its DNA target that explains the expression of the structural gene for resistance. To understand the loss of the high DNA affinity of the truncated repressor, we have determined the different dissociation constants of the system and solved the solution structure of the B. licheniformis monomeric repressor complexed to the semi-operating sequence OP1 of blaP (1/2OP1blaP) by using a de novo docking approach based on inter-molecular nuclear Overhauser effects and chemical-shift differences measured on each macromolecular partner. Although the N-terminal domain of the repressor is not subject to internal structural rearrangements upon DNA binding, the molecules adopt a tertiary conformation different from the crystallographic operator-repressor dimer complex, leading to a 30 degrees rotation of the monomer with respect to a central axis extended across the DNA. These results open new insights for the repression and induction mechanisms of bacterial resistance to beta-lactams.

The kinetic properties of the carboxy terminal domain of the Bacillus licheniformis 749/I BlaR penicillin-receptor shed a new light on the derepression of beta-lactamase synthesis.

To study the properties of the BlaR penicillin-receptor involved in the induction of the Bacillus licheniformisbeta-lactamase, the water-soluble carboxy terminal domain of the protein (BlaR-CTD) was overproduced in the periplasm of Escherichia coli JM105 and purified to protein homogeneity. Its interactions with various beta-lactam antibiotics were studied. The second-order acylation rate constants k2/K' ranged from 0.0017 to more than 1 micro M-1s-1 and the deacylation rate constants were lower than 4 x 10-5 s-1. These values imply a rapid to very rapid formation of a stable acylated adduct. BlaR-CTD is thus one of the most sensitive penicillin-binding proteins presently described. In the light of these results, the kinetics of beta-lactamase induction in Bacillus licheniformis were re-examined. When starting with a rather high cell density, a good beta-lactamase substrate such as benzylpenicillin is too sensitive to beta-lactamase-mediated hydrolysis to allow full induction. By contrast, a poor beta-lactamase substrate (7-aminocephalosporanic acid) can fully derepress beta-lactamase expression under conditions where interference of the antibiotic with cell growth is observed. These results suggest that acylation of the penicillin receptor is a necessary, but not sufficient, condition for full induction.

Advantages and drawbacks of nanospray for studying noncovalent protein-DNA complexes by mass spectrometry.

The noncovalent complexes between the BlaI protein dimer (wild-type and GM2 mutant) and its double-stranded DNA operator were studied by nanospray mass spectrometry and tandem mass spectrometry (MS/MS). Reproducibility problems in the nanospray single-stage mass spectra are emphasized. The relative intensities depend greatly on the shape of the capillary tip and on the capillary-cone distance. This results in difficulties in assessing the relative stabilities of the complexes simply from MS(1) spectra of protein-DNA mixtures. Competition experiments using MS/MS are a better approach to determine relative binding affinities. A competition between histidine-tagged BlaIWT (BlaIWTHis) and the GM2 mutant revealed that the two proteins have similar affinities for the DNA operator, and that they co-dimerize to form heterocomplexes. The low sample consumption of nanospray allows MS/MS spectra to be recorded at different collision energies for different charge states with 1 microL of sample. The MS/MS experiments on the dimers reveal that the GM2 dimer is more kinetically stable in the gas phase than the wild-type dimer. The MS/MS experiments on the complexes shows that the two proteins require the same collision energy to dissociate from the complex. This indicates that the rate-limiting step in the monomer loss from the protein-DNA complex arises from the breaking of the protein-DNA interface rather than the protein-protein interface. The dissociation of the protein-DNA complex proceeds by the loss of a highly charged monomer (carrying about two-thirds of the total charge and one-third of the total mass). MS/MS experiments on a heterocomplex also show that the two proteins BlaIWTHis and BlaIGM2 have slightly different charge distributions in the fragments. This emphasizes the need for better understanding the dissociation mechanisms of biomolecular complexes.