Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has emerged as a reliable tool for bacterial identification. This study compared the Bruker MALDI-TOF ...BioTyper MS (MBT) and 16S rRNA gene sequencing for the identification of Actinomyces and Actinotignum spp. The MBT identified 68/77 (88.3%) of Actinomyces isolates to the genus-level and 44/77 (57.1%) of Actinomyces isolates to the species-level using the manufacturer's identification criteria. The MBT did not yield reliable identification for only 1/77 (1.3%) and generated no identification for 8/77 (10.4%) of the isolates. No misidentifications were found. Discordance at the species level was observed for eight isolates. Overall, the MBT demonstrated good concordance with the 16S rRNA gene sequencing with the exception of the closely related species A. naeslundii, A. viscosus and A. oris. A variety of Actinomyces spp. were isolated from orocervicofacial/dental specimens, but only a limited number of species were isolated from urine or intra-abdominal specimens. This study confirms the utility of MBT in the identification of Actinomyces spp. and describes the diversity and anatomic niche of species in human clinical specimens from various body sites.
•MBT using direct transfer method provides rapid, inexpensive and accurate identification of Actinomyces spp.•Highlights the potential of MBT in the speciation of closely related species A. naeslundii, A. viscosus and A. oris.•MALDI-TOF MS facilitates the increase in knowledge about the distribution of Actinomyces spp. and their clinical relevance.
Export of cell surface pilins in Gram-positive bacteria likely occurs by the translocation of unfolded precursor polypeptides; however, how the unfolded pilins gain their native conformation is ...presently unknown. Here, we present physiological studies to demonstrate that the FimA pilin of Actinomyces oris contains two disulfide bonds. Alanine substitution of cysteine residues forming the C-terminal disulfide bridge abrogates pilus assembly, in turn eliminating biofilm formation and polymicrobial interaction. Transposon mutagenesis of A. oris yielded a mutant defective in adherence to Streptococcus oralis, and revealed the essential role of a vitamin K epoxide reductase (VKOR) gene in pilus assembly. Targeted deletion of vkor results in the same defects, which are rescued by ectopic expression of VKOR, but not a mutant containing an alanine substitution in its conserved CXXC motif. Depletion of mdbA, which encodes a membrane-bound thiol-disulfide oxidoreductase, abrogates pilus assembly and alters cell morphology. Remarkably, overexpression of MdbA or a counterpart from Corynebacterium diphtheriae, rescues the Δvkor mutant. By alkylation assays, we demonstrate that VKOR is required for MdbA reoxidation. Furthermore, crystallographic studies reveal that A. oris MdbA harbors a thioredoxin-like fold with the conserved CXXC active site. Consistently, each MdbA enzyme catalyzes proper disulfide bond formation within FimA in vitro that requires the catalytic CXXC motif. Because the majority of signal peptide-containing proteins encoded by A. oris possess multiple Cys residues, we propose that MdbA and VKOR constitute a major folding machine for the secretome of this organism. This oxidative protein folding pathway may be a common feature in Actinobacteria.
Background: Gram-positive bacteria secrete pilins through the Sec translocon in unfolded states.
Results: Disruption of pilus disulfide bonds or genetic disruption of oxidoreductase-encoding genes mdbA and vkor abrogates pilus assembly in Actinomyces oris.
Conclusion: MdbA and VKOR constitute a disulfide bond-forming machine in A. oris.
Significance: Oxidative protein folding may be common in Actinobacteria and an attractive target for antimicrobials.
Assembly of pili on the gram-positive bacterial cell wall involves 2 conserved transpeptidase enzymes named sortases: One for polymerization of pilin subunits and another for anchoring pili to ...peptidoglycan. How this machine controls pilus length and whether pilus length is critical for cell-to-cell interactions remain unknown. We report here in Actinomyces oris, a key colonizer in the development of oral biofilms, that genetic disruption of its housekeeping sortase SrtA generates exceedingly long pili, catalyzed by its pilus-specific sortase SrtC2 that possesses both pilus polymerization and cell wall anchoring functions. Remarkably, the srtA-deficient mutant fails to mediate interspecies interactions, or coaggregation, even though the coaggregation factor CafA is present at the pilus tip. Increasing ectopic expression of srtA in the mutant progressively shortens pilus length and restores coaggregation accordingly, while elevated levels of shaft pilins and SrtC2 produce long pili and block coaggregation by SrtA⁺ bacteria. With structural studies, we uncovered 2 key structural elements in SrtA that partake in recognition of pilin substrates and regulate pilus length by inducing the capture and transfer of pilus polymers to the cell wall. Evidently, coaggregation requires proper positioning of the tip adhesin CafA via modulation of pilus length by the housekeeping sortase SrtA.
The most common cause of empyema is a complicated parapneumonic effusion, but other foci of infection may also spread to the pleural space. A man in his early 30s with a history of testicular mixed ...germ cell tumor presented with a week of pleuritic chest pain. On admission, he was tachypneic, tachycardic, and had tested positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Imaging revealed a loculated large left pleural effusion with collapse of the left lung. A pigtail catheter was inserted, and the pleural effusion was evacuated. Fluid analysis indicated infection with
Actinomyces meyeri.
Clinical exam and mandible radiography ruled out infectious facial involvement. Intravenous ampicillin was started, but two days later the patient requested that the chest tube be removed, and he left the hospital against medical advice. The patient followed-up in our clinic one month later with a significantly improved medical condition. Treatment with oral amoxicillin-clavulanate for twelve months was prescribed.
Abstract Introduction To date, a variety of microbial species have been isolated from endodontic infections. However, endodontic clinical bacterial isolates have not been sufficiently characterized ...with regard to their capacity for antibiotic resistance and biofilm formation. In this study, antibiotic resistance and biofilm formation of 47 different aerobic and anaerobic bacterial isolates, belonging to 32 different species previously isolated from infected filled root canals, were studied. Methods Antibiotic sensitivity to 11 antibiotics including penicillin G, amoxicillin, clindamycin, gentamicin, vancomycin, tetracycline, doxycycline, fosfomycin, rifampicin, ciprofloxacin, and moxifloxacin was tested using the standardized Etest method (Bio Merieux, Marcy-1'Etoile, France). The antibiotic sensitivity of 4 control strains was also estimated in parallel. Additionally, the capacity to form biofilms was quantified using the microtiter plate test. Results Different aerobic and anaerobic bacterial species were either resistant against a number of antibiotics or showed high minimal inhibitory concentrations against clinically relevant antibiotics. Five aerobic and 2 anaerobic isolates, including Enterococcus faecalis , Streptococcus mutans , Lactobacillus fermentum , Actinomyces naeslundii , Actinomyces viscosus , Prevotella buccae , and Propionibacterium acidifaciens , were characterized as being high biofilm producers, whereas 8 aerobic and 3 anaerobic isolates were found to be moderate biofilm producers. Most isolates with resistance or markedly high minimal inhibitory concentration values were also either moderate biofilm producers or high biofilm producers. Conclusions These results suggest that the clinical significance of endodontic infections could include that they serve as a reservoir for antibiotic resistance. Furthermore, endodontic treatment should consider the adhesion and biofilm formation by a variety of bacteria.
Actinomycosis Wong, V K; Turmezei, T D; Weston, V C
BMJ,
10/2011, Letnik:
343, Številka:
7827
Journal Article
Recenzirano
SUMMARY POINTS Although rare, a high level of clinical suspicion is needed to diagnose and cure actinomycosis in patients with indolent, unresolving, or relapsing chronic inflammatory disease ...Actinomyces are commensals that become pathogenic when the mucosa is breached, and co-infection with other organisms is common Disease is defined by anatomical location; orecervicofacial disease is the most common, followed by thoracic and abdominopelvic disease A mass characteristically enlarges across tissue planes and local tissue invasion may lead to the formation of sinus tracts that can spontaneously heal and recur Actinomycosis often mimics other infections and malignancy—clinically and radiotogically It is generally treated with long term antibiotics, usually penicillin, but surgery may be needed
Sortase enzymes are attractive antivirulence drug targets that attach virulence factors to the surface of Staphylococcus aureus and other medically significant bacterial pathogens. Prior efforts to ...discover a useful sortase inhibitor have relied upon an in vitro activity assay in which the enzyme is removed from its native site on the bacterial surface and truncated to improve solubility. To discover inhibitors that are effective in inactivating sortases in vivo, we developed and implemented a novel cell-based screen using Actinomyces oris, a key colonizer in the development of oral biofilms. A. oris is unique because it exhibits sortase-dependent growth in cell culture, providing a robust phenotype for high throughput screening (HTS). Three molecules representing two unique scaffolds were discovered by HTS and disrupt surface protein display in intact cells and inhibit enzyme activity in vitro. This represents the first HTS for sortase inhibitors that relies on the simple metric of cellular growth and suggests that A. oris may be a useful platform for discovery efforts targeting sortase.
Recent advancements in DNA-based approaches have led to the identification of uncommon and rare bacterial pathogens. In this study, by utilizing a DNA-based approach, a total of 1043 clinical ...specimens were processed for the identification of actinobacteria targeting the 16S rRNA and gyrB genes. Drug susceptibility testing was also conducted using micro-broth dilution and PCR. Two isolates of Nocardia flavorosea and Rhodococcus erythropolis were reported for the first time in Iran. Also, Nocardiopsis dassonvillei, Streptomyces olivaceus, and Streptomyces griseus were reported for the first time in Asia. Infections caused by Nocardia caishijiensis and Prauserella muralis have also been reported in this study. The first Asian case of pulmonary infection caused by Nocardia ignorata and the first global case of brain abscess caused by Nocardia ninae and Nocardia neocaledoniensis have been reported in this study. Overall 30 isolates belonging to 6 genera (Nocardia, Streptomyces, Rodoccoccus, Nocardiopsis, Rothia, and Prauserella) were detected in 30 patients. All 30 isolates were susceptible to amikacin and linezolid. Three isolates including Nocardia otitidiscaviarum (n = 2) and Nocardia flavorosea (n = 1) were resistant to trimethoprim-sulfamethoxazole which were the first trimethoprim-sulfamethoxazole resistant clinical actinomycetes in Iran. Isolation of rare species of actinomycetes particularly Nocardia spp. requires urgent action before they spread clinically particularly among immunocompromised patients.
Dental caries (tooth decay) is an infectious disease. Its etiology is not fully understood from the microbiological perspective. This study characterizes the diversity of microbial flora in the ...saliva of children with and without dental caries. Children (3-4 years old) with caries (
= 20) and without caries (
= 20) were recruited. Unstimulated saliva (2 mL) was collected from each child and the total microbial genomic DNA was extracted. DNA amplicons of the V3-V4 hypervariable region of the bacterial 16S rRNA gene were generated and subjected to Illumina Miseq sequencing. A total of 17 phyla, 26 classes, 40 orders, 80 families, 151 genera, and 310 bacterial species were represented in the saliva samples. There was no significant difference in the microbiome diversity between caries-affected and caries-free children (
> 0.05). The relative abundance of several species (
,
,
sp.
,
, and
) was higher in the caries-affected group than in the caries-free group (
< 0.05).
and
sp.
were more abundant in caries-free children than in caries-affected children (
< 0.05). The salivary microbiome profiles of caries-free and caries-affected children were similar. Salivary counts of certain bacteria such as
and
may be useful for screening/assessing children's risk of developing caries.
Summary
Sortase, a cysteine‐transpeptidase conserved in Gram‐positive bacteria, anchors on the cell wall many surface proteins that facilitate bacterial pathogenesis and fitness. Genetic disruption ...of the housekeeping sortase in several Gram‐positive pathogens reported thus far attenuates virulence, but not bacterial growth. Paradoxically, we discovered that depletion of the housekeeping sortase SrtA was lethal for Actinomyces oris; yet, all of its predicted cell wall‐anchored protein substrates (AcaA‐N) were individually dispensable for cell viability. Using Tn5‐transposon mutagenesis to identify factors that upend lethality of srtA deletion, we uncovered a set of genetic suppressors harbouring transposon insertions within genes of a locus encoding AcaC and a LytR‐CpsA‐Psr (LCP)‐like protein. AcaC was shown to be highly glycosylated and dependent on LCP for its glycosylation. Upon SrtA depletion, the glycosylated form of AcaC, hereby renamed GspA, was accumulated in the membrane. Overexpression of GspA in a mutant lacking gspA and srtA was lethal; conversely, cells overexpressing a GspA mutant missing a membrane‐localization domain were viable. The results reveal a unique glycosylation pathway in A. oris that is coupled to cell wall anchoring catalysed by sortase SrtA. Significantly, this novel phenomenon of glyco‐stress provides convenient cell‐based assays for developing a new class of inhibitors against Gram‐positive pathogens.
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