Whole genome-based antimicrobial resistance, virulence, and phylogenetic characteristics of Trueperella pyogenes clinical isolates from humans and animals.

Trueperella pyogenes is an opportunistic zoonotic bacterial pathogen, whose antimicrobial resistance, virulence, and genetic relatedness between strains from animals and humans are barely studied. These characteristics were therefore analyzed for clinical T. pyogenes strains from 31 animals of 11 different species and 8 humans determining their complete circular genome sequence and antimicrobial susceptibility. The MICs of 19 antimicrobials including 3 antiseptics correlated to the resistance genes identified in silico within the genomes revealing a predominance of resistance to streptomycin (aadA9), sulfamethoxazole (sul1), and tetracycline (tet(33), tet(W/N/W)) among strains from humans and cattle. Additional resistance genes (erm(X), erm(56), cmx, drfA1, aadA1, aph(3'')-Ib (strA), aph(6)-Id (strB), aac(3)-IVa, aph(4)-Ia) were found only sporadically. The resistance genes were localized on genetic elements integrated into the chromosome. A cgMLST-based phylogenetic analysis revealed two major clusters each containing genetically diverse strains. The human strains showed the closest relatedness to strains from cattle. Virulence genes coding for fimbriae (fimA, fimC), neuroamidase (nanP, nanH), pyolysin (plo), and collagen binding protein (cbpA) were identified in strains from different hosts, but no correlation was observed between virulence factors and strain origin. The existence of resistance genes typically found in Gram-negative bacteria within the Gram-positive T. pyogenes indicates a wider capacity to adapt to antimicrobial selective pressure. Moreover, the presence of similar antimicrobial resistance profiles found in cattle and human strains as well as their closest relatedness suggests common zoonotic features and cattle as the potential source for human infections.

Clinical endometritis with Trueperella pyogenes reduces reproductive performance and milk production in dairy cows.

The aim of this study was to identify the impact of Trueperella pyogenes in cows with clinical endometritis (CE) on reproductive performance and milk production in affected cows. In total, 230 lactating Holstein dairy cows from six commercial dairy herds were sampled once between 28 and 33 days post-partum. Cows included in the present study did not receive antibiotic or anti-inflammatory treatments prior to the experimental period. Clinical endometritis (CE) was characterized as cow with vaginal mucus score = 3 (>50% of purulent vaginal discharge) and >18% polymorphonuclear neutrophilic leukocyte (PMNL). The body condition scores (BCS) and milk production were evaluated at the time of enrolment. The identification of isolated bacteria was carried out through the analysis of MALDI-TOF MS (matrix-assisted laser desorption ionization time-of-flight mass spectrometry). According to uterine health, three groups of dairy cows were formed: healthy control cows without T. pyogenes (n = 147), CE cows with T. pyogenes (n = 22) and CE cows without T. pyogenes (n = 61). CE cows with T. pyogenes had lower BCS, milk production and conception at first AI (p < .01) than CE cows without T. pyogenes and control cows. Furthermore, CE cows with T. pyogenes had higher (p < .01) service per pregnancy and had greater (p < .01) days to get pregnant than CE cows without T. pyogenes and control cows. This study demonstrates that CE cows with T. pyogenes had impaired reproductive performance and milk production when compared to cows without CE and CE cows without T. pyogenes. This information can contribute to a strategic treatment in cows affected by clinical endometritis, favouring the rational use of antibiotics on dairy farms.

Trueperella pyogenes pyolysin inhibits lipopolysaccharide-induced inflammatory response in endometrium stromal cells via autophagy- and ATF6-dependent mechanism.

Trueperella pyogenes (T. pyogenes) is a common opportunistic pathogen of many livestock and play an important regulation role during multibacterial infection and interaction with the host by its primary virulence factor pyolysin (PLO). The purpose of this study was to investigate the regulation role of PLO which serve as a combinational pathogen with lipopolysaccharide (LPS) during endometritis. In this study, the expression of bioactive recombinant PLO (rPLO) in a prokaryotic expression system and its purification are described. Moreover, we observed that rPLO inhibited the innate immune response triggered by LPS and that methyl-ß-cyclodextrin (MBCD) abrogated this inhibitory effect in goat endometrium stromal cells (gESCs). Additionally, we show from pharmacological and genetic studies that rPLO-induced autophagy represses gene expression by inhibiting NLRP3 inflammasome activation. Importantly, this study reported that ATF6 serves as a primary regulator of the cellular inflammatory reaction to rPLO. Overall, these observations suggest that T. pyogenes PLO could create an immunosuppressive environment for other pathogens invasion by regulating cellular signaling pathways.

A Case of Brain Abscess Caused by Actinomyces Cardiffensis and Parvimonas Micra.

Brain abscesses occur in 0.3-1.3 per 100,000 worldwide each year with 0.4-0.9 in Japan alone. Most of the causes are direct infection from a nearby infectious lesion and are rarely caused by an odontogenic infection. Here, we reported a case of brain abscess suspected to be associated with odontogenic infection. The patient was a 55-year-old woman. Blurred eyes and pain in the left eye noted, for which she consulted an ophthalmologist, but her eyes were normal. She was conscious and was able to converse clearly, but she could not read the letters and had difficulty in writing at the time of admission. A brain abscess was diagnosed based on the head magnetic resonance imaging (MRI) and clinical course, and a small craniotomy abscess drainage was performed. A. cardiffensis and P. micra were detected in the abscess, suggesting the involvement of periodontal disease bacteria. After the surgery, antimicrobial treatment was performed for about 2 months. At the same time, perioperative treatment was performed. On the 70th day after the surgery, tooth extraction, which was considered as the source of infection, was performed. The patient was discharged 74 days after surgery. A good turning point was obtained without relapse of symptoms.

Differences in phenotypic and genetic characteristics of Trueperella pyogenes detected in slaughtered cattle and pigs with septicemia.

We investigated the hemolytic properties, biochemical properties, and possession of virulence factor genes of Trueperella pyogenes isolated from cattle and pigs with septicemia. The porcine strains showed significantly stronger hemolyticity than the bovine strains. In addition, T. pyogenes from cattle and pigs also differed in biochemical properties. Virulence factor genes (nanP, cbpA, fimC, and fimE) were more prevalent in bovine strains, whereas other virulence factor genes (nanH and fimG) were more prevalent in porcine strains. T. pyogenes isolated from pig and cattle with septis cases in Japanese meat inspection showed variability in biochemical and genetic properties. Differences were observed between porcine and bovine strain in term of the hemolytic strength and possession of genes for factors promoting adhesions which are considered pathogenic.

Pathogenicity and Virulence of Trueperella pyogenes: A Review.

Bacteria from the species Trueperella pyogenes are a part of the biota of skin and mucous membranes of the upper respiratory, gastrointestinal, or urogenital tracts of animals, but also, opportunistic pathogens. T. pyogenes causes a variety of purulent infections, such as metritis, mastitis, pneumonia, and abscesses, which, in livestock breeding, generate significant economic losses. Although this species has been known for a long time, many questions concerning the mechanisms of infection pathogenesis, as well as reservoirs and routes of transmission of bacteria, remain poorly understood. Pyolysin is a major known virulence factor of T. pyogenes that belongs to the family of cholesterol-dependent cytolysins. Its cytolytic activity is associated with transmembrane pore formation. Other putative virulence factors, including neuraminidases, extracellular matrix-binding proteins, fimbriae, and biofilm formation ability, contribute to the adhesion and colonization of the host tissues. However, data about the pathogen-host interactions that may be involved in the development of T. pyogenes infection are still limited. The aim of this review is to present the current knowledge about the pathogenic potential and virulence of T. pyogenes.

Use of MALDI-TOF MS in diagnostic microbiology for identification of species that conventional methods usually fail to identify

(MALDI-TOF MS) has been used in clinical diagnostic laboratories for the identification of microorganisms. It has a relevant advantage compared to other methods in terms of speed to provide results, being an alternative for addressing restrictions in clinical diagnosis as it may replace or complement existing identification techniques. This is especially important because some rare microorganisms would be identified only by higher cost techniques which are not widely available, such as genetic sequencing. Thus, the present paper reports two cases in which uncommon microorganisms were identified effectively and quickly. (AU)

Genomic characterisation, detection of genes encoding virulence factors and evaluation of antibiotic resistance of Trueperella pyogenes isolated from cattle with clinical metritis.

Trueperella pyogenes is one of the most important microorganisms causing metritis in post-partum cattle. Co-infection with other bacterial species such as Escherichia coli or Fusobacterium necrofurom increases the severity of the disease and the persistence of bacteria in utero. The aim of this study was to investigate the frequency of T. pyogenes strains, and their virulence and antimicrobial resistant profiles in metritis cases. The study was carried out on 200 samples obtained from metritis discharges of postpartum cattle on 18 farms around Tehran, Iran. Sixty-five T. pyogenes isolates (32.5%) were identified, of which 16 isolates were detected as pure cultures and the other 49 isolates from cultures most commonly mixed with E. coli or F. necrofurom. In terms of diversity in biochemical characteristic of T. pyogenes strains, 8 different biotypes were identified among the isolates. Single or multi antimicrobial resistance was observed in 48 isolates (73.9%), which was mostly against trimethoprim sulfamethoxazole, azithromycin, erythromycin and streptomycin. The tetracycline resistance gene tetW and macrolide resistance genes ermB and ermX were detected in 30, 18 and 25 isolates, respectively. In the screening of genes encoding virulence factors, fimA and plo genes were identified in all tested isolates. Genes encoding nanP, nanH, fimC, fimG, fimE and cbpA were detected in 50, 54, 45, 40, 50 and 37 of isolates, respectively. Thirteen different genotypes were observed in these T. pyogenes isolates. A significant association between clonal types and virulence factor genes, biochemical profile, CAMP test result, severity of the disease and sampling time was detected.

Replacing the 238th aspartic acid with an arginine impaired the oligomerization activity and inflammation-inducing property of pyolysin.

Trueperella pyogenes (T. pyogenes) is an important opportunistic pathogen. Pyolysin (PLO) importantly contributes to the pathogenicity of T. pyogenes. However, the relationship between the structure and function and the virulence of PLO is not well documented. In the current study, recombinant PLO (rPLO) and three rPLO mutants were prepared. rPLO D238R, a mutant with the 238th aspartic acid replaced with an arginine, showed impairment in oligomerization activity on cholesterol-containing liposome and pore-forming activity on sheep red blood cell membrane. Further study employing the prepared mutants confirmed that the pore-forming activity of PLO is essential for inducing excessive inflammation responses in mice by upregulating the expression levels of IL-1ß, TNF-α, and IL-6. By contrast, rPLO P499F, another mutant with impaired cell membrane binding capacity, elicited an inflammation response that was dependent on pathogen-associated molecular pattern (PAMP) activity, given that the mutant significantly upregulated the expression of IL-10 in macrophages and in mice, whereas rPLO did not. Results indicated that domain 1 of the PLO molecule plays an important role in maintaining pore-forming activity. Moreover, the PLO pore-forming activity and not PAMP activity is responsible for the inflammation-inducing effect of PLO. The results of this study provided new information for research field on the structure, function, and virulence of PLO. ABBREVIATIONS: T. pyogenes: Trueperella pyogenes; PLO: Pyolysin; rPLO: recombinant PLO; PAMP: pathogen-associated molecular pattern; CDCs: cholesterol-dependent cytolysins; PLY: pneumolysin; NLRP3: NLR family pyrin domain containing protein 3; PRRs: pattern recognition receptors; Asp: aspartic acid; TLR4: Toll-like receptor 4; Arg: arginine; Asn: asparagine; IPTG: Isopropyl-ß-d-thiogalactoside; PBS: phosphate-buffered saline; sRBCs: sheep red blood cells; TEM: Transmission electron microscopy; RBCM: red blood cell membrane; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; NC membrane: nitrocellulose membrane; SDS-AGE: dodecyl sulfate agarose gel electrophoresis; MDBK cells: Madin-Darby bovine kidney cells; RPMI-1640 medium: Roswell Park Memorial Institute-1640 medium; FBS: fetal bovine serum; BMDMs: bone marrow-derived macrophages; TNF-α: tumor necrosis factor α; IL-1ß: interleukin-1ß; IFN-γ: interferon-γ; TGF-ß: transforming growth factor-ß; ELISA: enzyme-linked immunosorbent assay.

A HEADACHE FROM OUR PAST? INTRACRANIAL ABSCESS DISEASE, VIRULENCE FACTORS OF TRUEPERELLA PYOGENES, AND A LEGACY OF TRANSLOCATING WHITE-TAILED DEER ( ODOCOILEUS VIRGINIANUS).

Trueperella pyogenes, a bacterial opportunistic pathogen residing along the skin layer of apparently healthy animals, is the etiologic agent of intracranial abscessation-suppurative meningoencephalitis, a cause of mortality for male white-tailed deer ( Odocoileus virginianus). Occurrence of this disease has been speculated to be influenced by virulence of T. pyogenes residing on white-tailed deer in geographically distinct metapopulations. To determine if differences in virulence potential of T. pyogenes could affect occurrence of disease across populations, we examined if frequency of seven virulence genes of T. pyogenes from forehead swabs of 186 apparently healthy white-tailed deer differed between sites in the state of Georgia, US, where ≥1 male tested positive for a cranial abscess and sites where no individuals tested positive for a cranial abscess. We detected six of seven virulence genes more frequently at sites where we detected ≥1 male with a cranial abscess compared to sites where we did not detect any individuals with a cranial abscess ( nanH, P<0.001; nanP, P=0.007; fimA, P<0.001; fimC, P=0.037; fimE, P<0.009; fimG, P<0.001; and cbpA, P=0.872). Our findings suggest differences in the pathogenic potential of T. pyogenes at individual sites may help to explain spatial variability of this disease. Anecdotally, the incidence of cranial abscess disease in Georgia seems to be associated with areas that were restocked with white-tailed deer from a high-fenced property in Wisconsin, US. Given the spatial distribution of this disease, we speculate that these genetic differences in T. pyogenes may have arisen from white-tailed deer restocking efforts, and our observations may be a legacy of an introduced disease manifesting itself generations later.

Quorum-sensing molecules N-acyl homoserine lactones inhibit Trueperella pyogenes infection in mouse model.

Trueperella pyogenes is a gram-positive opportunistic pathogen normally causes mastitis, liver abscesses and pneumonia of economically important livestock. It has been suggested that gram-negative bacteria can suppress the growth and virulence of T. pyogenes in vitro by using the quorum-sensing (QS) signal molecules and cause the transition of predominant species. However, whether these QS signals can be used as potential anti-virulence drugs against T. pyogenes infection is unclear. In this study, the in vivo inhibitory effect N-acyl homoserine lactones (AHLs) from Escherichia coli and Pseudomonas aeruginosa on T. pyogenes was tested by using mouse model. Mice were first peritoneally infected with T. pyogenes followed by intravenous injection of N-Octanoyl-DL-homoserine lactone (C8HSL) or N-(3-oxododecanoyl) homoserine-l-lactone (C12HSL). The results showed that C8HSL and C12HSL significantly reduced bacterial load and increased the survival rate of mice against T. pyogenes challenge. Additionally, the treatment of AHLs promoted the secretion of IL-1ß, IL-6, IL-8 and TNF-α in mouse peritoneal fluid, and significantly decreased the expression levels of virulence genes of residual T. pyogenes. Importantly, murine macrophages rapidly phagocytosed bacteria when they were treated with AHLs compared to untreated cells. Collectively, our findings provide a major advance in understanding the inhibitory effect of AHLs in vivo and a promise for developing new clinical or veterinary treatments of T. pyogenes-related infection.

Different inflammatory responses of bovine oviductal epithelial cells in vitro to bacterial species with distinct pathogenicity characteristics and passage number.

The bovine oviduct provides the site for fertilization and early embryonic development. Modifications to this physiological environment, for instance the presence of pathogenic bacterial species, could diminish reproductive success at early stages of pregnancy. The aim of this study was to elucidate the inflammatory responses of bovine oviductal epithelial cells (BOEC) to a pathogenic bacterial species (Trueperella pyogenes) and a potentially pathogenic bacterium (Bacillus pumilus). BOEC from four healthy animals were isolated, cultured in passage 0 (P0) and passaged until P3. Trypan blue staining determined BOEC viability during 24 h co-culture with different multiplicities of infection (MOI) of T. pyogenes (MOI 0.01, 0.05, 0.1 and 1) or B. pumilus (MOI 1 and 10). BOEC remained viable when co-cultured with T. pyogenes at MOI 0.01 and with B. pumilus at MOI 1 and 10. Extracted total RNA from control and bacteria co-cultured samples was subjected to reverse transcription-quantitative polymerase chain reaction (RTq-PCR) to determine mRNA expression of various studied genes. The rate of release of interleukin 8 (IL8) and prostaglandin E2 (PGE2) from BOEC was measured by ELISA after 24 h co-culture with bacteria. RT-qPCR of various selected pro-inflammatory factors revealed similar mRNA expression of pro-inflammatory factors in BOEC co-cultured with T. pyogenes and in the controls. Higher mRNA expression of IL 1A, -1B, tumor necrosis factor alpha and CXC ligand (CXCL) 1/2, -3, -5 and IL8 and PG synthesis enzymes in BOEC co-cultured with B. pumilus was observed. In the presence of B. pumilus a higher amount of IL8 and PGE2 was released from BOEC than from controls. The viability and pro-inflammatory response of P3 BOEC incubated with bacteria was lower than in P0 BOEC. These findings illustrate the pathogenicity of T. pyogenes towards BOEC in detail and the potential role of B. pumilus in generating inflammation in oviductal cells. Culturing conditions influenced the pro-inflammatory responses of BOEC towards bacteria. Therefore, researchers conducting epithelial-bacterial in vitro co-culture should not underestimate the effects of these parameters.

Draft genome sequence of Actinotignum schaalii DSM 15541T: Genetic insights into the lifestyle, cell fitness and virulence.

The permanent draft genome sequence of Actinotignum schaalii DSM 15541T is presented. The annotated genome includes 2,130,987 bp, with 1777 protein-coding and 58 rRNA-coding genes. Genome sequence analysis revealed absence of genes encoding for: components of the PTS systems, enzymes of the TCA cycle, glyoxylate shunt and gluconeogensis. Genomic data revealed that A. schaalii is able to oxidize carbohydrates via glycolysis, the nonoxidative pentose phosphate and the Entner-Doudoroff pathways. Besides, the genome harbors genes encoding for enzymes involved in the conversion of pyruvate to lactate, acetate and ethanol, which are found to be the end products of carbohydrate fermentation. The genome contained the gene encoding Type I fatty acid synthase required for de novo FAS biosynthesis. The plsY and plsX genes encoding the acyltransferases necessary for phosphatidic acid biosynthesis were absent from the genome. The genome harbors genes encoding enzymes responsible for isoprene biosynthesis via the mevalonate (MVA) pathway. Genes encoding enzymes that confer resistance to reactive oxygen species (ROS) were identified. In addition, A. schaalii harbors genes that protect the genome against viral infections. These include restriction-modification (RM) systems, type II toxin-antitoxin (TA), CRISPR-Cas and abortive infection system. A. schaalii genome also encodes several virulence factors that contribute to adhesion and internalization of this pathogen such as the tad genes encoding proteins required for pili assembly, the nanI gene encoding exo-alpha-sialidase, genes encoding heat shock proteins and genes encoding type VII secretion system. These features are consistent with anaerobic and pathogenic lifestyles. Finally, resistance to ciprofloxacin occurs by mutation in chromosomal genes that encode the subunits of DNA-gyrase (GyrA) and topisomerase IV (ParC) enzymes, while resistant to metronidazole was due to the frxA gene, which encodes NADPH-flavin oxidoreductase.

Identification of B-cell linear epitopes in domains 1-3 of pyolysin of Trueperella pyogenes using polyclonal antibodies.

Trueperella pyogenes is an important opportunistic pathogen. Pyolysin (PLO) makes important contributions to the pathogenicity of T. pyogenes. However, the structure and function of PLO has not been well documented. In the current study, epitopes in domain 1-3 of PLO have been mapped using rabbit anti-recombinant PLO (rPLO) polyclonal antibodies, and then the results were re-checked by using mouse and chicken anti-rPLO polyclonal antibodies, respectively. The results indicated that the region of aa 281-393 in PLO could not elicit antibodies against linear epitopes. A total of six B cell linear epitopes have been found in domain 1 of PLO. Two of the six epitopes (EP1 and EP2) were used to immunize mice and chicken. Chicken anti-EP1 and anti-EP2 serum and mouse anti-EP2 serum could react with rPLO and corresponding epitope polypeptide in western blot assay; however, only mouse anti-EP2 serum shows weak anti-hemolysis effect in the rPLO and sheep red blood system. Our results provide some new information to the research field of PLO structure and function.

Bovine Endometrial Epithelial Cells Scale Their Pro-inflammatory Response In vitro to Pathogenic Trueperella pyogenes Isolated from the Bovine Uterus in a Strain-Specific Manner.

Among different bacteria colonizing the bovine uterus, Trueperella pyogenes is found to be associated with clinical endometritis (CE). The ability of cows to defend against T. pyogenes infections depends on the virulence of invading bacteria and on the host's innate immunity. Therefore, to gain insights into bacterial factors contributing to the interplay of this host pathogen, two strains of T. pyogenes were included in this study: one strain (TP2) was isolated from the uterus of a postpartum dairy cow developing CE and a second strain (TP5) was isolated from a uterus of a healthy cow. The two strains were compared in terms of their metabolic fingerprints, growth rate, virulence gene transcription, and effect on bovine endometrial epithelial cells in vitro. In addition, the effect of the presence of peripheral blood mononuclear cells (PBMCs) on the response of endometrial epithelial cells was evaluated. TP2, the strain isolated from the diseased cow, showed a higher growth rate, expressed more virulence factors (cbpA, nanH, fimE, and fimG), and elicited a higher mRNA expression of pro-inflammatory factors (PTGS2, CXCL3, and IL8) in bovine endometrial epithelial cells compared with TP5, the strain isolated from the healthy cow. The presence of PBMCs amplified the mRNA expression of pro-inflammatory factors (PTGS2, CXCL3, IL1A, IL6, and IL8) in bovine endometrial epithelial cells co-cultured with live TP2 compared with untreated cells, especially as early as after 4 h. In conclusion, particular strain characteristics of T. pyogenes were found to be important for the development of CE. Furthermore, immune cells attracted to the site of infection might also play an important role in up-regulation of the pro-inflammatory response in the bovine uterus and thus significantly contribute to the host-pathogen interaction.

Comparative transcriptome analysis of Trueperella pyogenes reveals a novel antimicrobial strategy.

Trueperella pyogenes is a prevalent opportunistic bacterium that normally causes diverse suppurative lesions, endometritis and pneumonia in various economically important animals. Although the genomic information of this species has been announced, little is known about its functional profiles. In this study, by performing a comparative transcriptome analysis between the highly and moderately virulent T. pyogenes isolates, we found the expression of a LuxR-type DNA-binding response regulator, PloR, was significantly up-regulated in the highly virulent T. pyogenes. Protein crystal structure prediction and primary functional assessment suggested that, the quorum-sensing signal molecules of Gram-negative bacteria such as Pseudomonas aeruginosa and Escherichia coli could significantly inhibit the growth, biofilm production and hemolysis of T. pyogenes by binding to the upstream sensor histidine kinase, PloS. Therefore, the PloS/PlosR two-component regulatory system might dominate the virulence of T. pyogenes. Our findings provide a major advance in understanding the pathogenesis of T. pyogenes, and may shed new light on the development of novel therapeutic strategies to control T. pyogenes infection.

Actinotignum schaalii (formerly Actinobaculum schaalii): a newly recognized pathogen-review of the literature.

The genus Actinotignum contains three species, Actinotignum schaalii (formerly Actinobaculum schaalii), Actinotignum urinale and Actinotignum sanguinis. A. schaalii is the species most frequently involved in human infections, with 172 cases, mostly urinary tract infections (UTIs), reported so far. Invasive infections have also been described. This facultative anaerobic Gram-positive rod is part of the urinary microbiota of healthy patients. It is responsible for UTIs, particularly in elderly men and young children. A. schaalii is an underestimated cause of UTIs because of its fastidious growth on usual media and difficulties associated with its identification using phenotypic methods. Indeed, this slow-growth bacterium requires blood-enriched media and an incubation time of 48 hours under anaerobic or 5% CO2 atmosphere. Furthermore, only matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) or molecular-based methods allow the accurate identification of this bacteria. MALDI-TOF using Microflex LT with the Biotyper database (Bruker Daltonics, Bremen, Germany) is the most reliable technology for the routine identification of A. schaalii. The identification of this uropathogen is all the more important because it is resistant to trimethoprim/sulfamethoxazole and second-generation quinolones that are widely used in the treatment of UTIs. Antimicrobial therapy using ß-lactams prolonged for up to 2 weeks is the most efficient treatment and should be recommended. Microbiologists should assess the presence of A. schaalii in urine using appropriate culture and identification methods in the case of a direct examination that is positive for small coccoid rods, a negative nitrite urinary stick associated with leukocyturia, treatment failure with trimethoprim/sulfamethoxazole or fluoroquinolones, or undocumented, repeated UTIs.

Protective role of the dynamin inhibitor Dynasore against the cholesterol-dependent cytolysin of Trueperella pyogenes.

The virulence of many Gram-positive bacteria depends on cholesterol-dependent cytolysins (CDCs), which form pores in eukaryotic cell plasma membranes. Pyolysin (PLO) from Trueperella pyogenes provided a unique opportunity to explore cellular responses to CDCs because it does not require thiol activation. Sublytic concentrations of PLO stimulated phosphorylation of MAPK ERK and p38 in primary stromal cells, and induced autophagy as determined by protein light-chain 3B cleavage. Although, inhibitors of MAPK or autophagy did not affect PLO-induced cytolysis. However, 10 µM 3-hydroxynaphthalene-2-carboxylic acid-(3,4-dihydroxybenzylidene)-hydrazide (Dynasore), a dynamin guanosine 5'-triphosphatase inhibitor, protected stromal cells against PLO-induced cytolysis as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (85 ± 17% versus 50 ± 9% cell viability), measuring extracellular ATP, and kinetic assays. This was a generalized mechanism because Dynasore also protected HeLa cells against streptolysin O. Furthermore, the effect was reversible, with stromal cell sensitivity to PLO restored within 30 minutes of Dynasore removal. The protective effect of Dynasore was not conferred by dynamin inhibition, induction of ERK phosphorylation, or Dynasore binding to PLO. Rather, Dynasore reduced cellular cholesterol and disrupted plasma membrane lipid rafts, similar to positive control methyl-ß-cyclodextrin. Dynasore is a tractable tool to explore the complexity of cholesterol homeostasis in eukaryotic cells and to develop strategies to counter CDCs.