Comprehensive lipidomic analysis of the genus Cutibacterium.

Cutibacterium are part of the human skin microbiota and are opportunistic microorganisms that become pathogenic in immunodeficient states. These lipophilic bacteria willingly inhabit areas of the skin where sebaceous glands are abundant; hence, there is a need to thoroughly understand their metabolism. Lipids are no longer considered only structural elements but also serve as signaling molecules and may have antigenic properties. Lipidomics remains a major research challenge, mainly due to the diverse physicochemical properties of lipids. Therefore, this study aimed to perform a large comparative lipidomic analysis of eight representatives of the Cutibacterium genus, including four phylotypes of C. acnes and two strains of C. granulosum, C. avidum, and C. namnetense. Lipidomic analysis was performed by liquid chromatography‒mass spectrometry (LC-MS) in both positive and negative ion modes, allowing the detection of the widest range of metabolites. Fatty acid analysis by gas chromatography‒mass spectrometry (GC-MS) corroborated the lipidomic data. As a result, 128 lipids were identified, among which it was possible to select marker compounds, some of which were characteristic even of individual C. acnes phylotypes. These include phosphatidylcholine PC 30:0, sphingomyelins (SM 33:1, SM 35:1), and phosphatidylglycerol with an alkyl ether substituent PG O-32:0. Moreover, cardiolipins and fatty acid amides were identified in Cutibacterium spp. for the first time. This comparative characterization of the cutibacterial lipidome with the search for specific molecular markers reveals its diagnostic potential for clinical microbiology. IMPORTANCE: Cutibacterium (previously Propionibacterium) represents an important part of the human skin microbiota, and its role in clinical microbiology is growing due to opportunistic infections. Lipidomics, apart from protein profiling, has the potential to prove to be a useful tool for defining the cellular fingerprint, allowing for precise differentiation of microorganisms. In this work, we presented a comparative analysis of lipids found in eight strains of the genus Cutibacterium, including a few C. acnes phylotypes. Our results are one of the first large-scale comprehensive studies regarding the bacterial lipidome, which also enabled the selection of C. acnes phylotype-specific lipid markers. The increased role of lipids not only as structural components but also as diagnostic markers or potential antigens has led to new lipid markers that can be used as diagnostic tools for clinical microbiology. We believe that the findings in our paper will appeal to a wide range of researchers.

Identification of environmental Actinobacteria in buildings by means of chemotaxonomy, 16S rRNA sequencing, and MALDI-TOF MS.

Actinobacteria are abundant in soil and other environmental ecosystems and are also an important part of the human microbiota. Hence, they can also be detected in indoor environments and on building materials, where actinobacterial proliferation on damp materials can indicate moisture damage. The aim of this study was to evaluate the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for the identification of 28 environmental strains of Actinobacteria isolated from building materials and indoor and outdoor air samples, mainly collected in the context of moisture damage investigations in buildings in Finland. The 16S rRNA gene sequencing and chemotaxonomic analyses were performed, and results were compared with the MALDI-TOF MS Biotyper identification. Using 16S rRNA gene sequencing, all isolates were identified on the species or genus level and were representatives of Streptomyces, Nocardia, and Pseudonocardia genera. Based on MALDI-TOF MS analysis, initially, 11 isolates were identified as Streptomyces spp. and 1 as Nocardia carnea with a high identification score. After an upgrade in the MALDI-TOF MS in-house database and re-evaluation of mass spectra, 13 additional isolates were identified as Nocardia, Pseudonocardia, and Streptomyces. MALDI-TOF MS has the potential in environmental strain identification; however, the standard database needs to be considerably enriched by environmental Actinobacteria representatives. IMPORTANCE: The manuscript addresses the challenges in identifying environmental bacteria using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) Biotyper-based protein profiling. The matter of the studies-actinobacterial strains-has been isolated mostly from building materials that originated from a confirmed moisture-damaged situation. Polyphasic taxonomy, 16S RNA gene sequencing, and MALDI-TOF mass spectrometry were applied for identification purposes. In this experimental paper, a few important facts are highlighted. First, Actinobacteria are abundant in the natural as well as built environment, and their identification on the species and genus levels is difficult and time-consuming. Second, MALDI-TOF MS is an effective tool for identifying bacterial environmental strains, and in parallel, continuous enrichment of the proteomics mass spectral databases is necessary for proper identification. Third, the chemical approach aids in the taxonomical inquiry of Actinobacteria environmental strains.

Cutaneous tuberculosis-ambiguous transmission, bacterial diversity with biofilm formation in humoral abnormality: case report illustration.

Background: Cutaneous tuberculosis (CTB) and its paucibacillary forms are rare and difficult to diagnose, especially in immunocompromised patients with significant comorbidity. The aim of the study was to introduce the modern concept of the microbiome and diagnostic chain into clinical practice (patient-centered care) with the presentation of an atypical form of cutaneous tuberculosis with necrotizing non-healing ulcers leading to polymicrobial infection. Methods: The study material included samples from sputum, broncho-alveolar lavage and skin ulcer, taken from a patient developing cutaneous tuberculosis. The microbiological investigation was performed, and identification of the isolates was carried out using genotyping and the matrix-assisted laser desorption ionization-time of flight mass spectrometry. Results: The immunocompromised patient with humoral abnormality (plasma cell dyscrasia) and severe paraproteinemia developed multiorgan tuberculosis. Although cutaneous manifestation preceded systemic and pulmonary symptoms (approximately half a year), the mycobacterial genotyping confirmed the same MTB strain existence in skin ulcers and the respiratory system. Therefore, the infectious chain: transmission, the portal of entry, and bacterial spreading in vivo, were unclear. Microbial diversity found in wound microbiota (among others Gordonia bronchialis, Corynebacterium tuberculostearicum, Staphylococcus haemolyticus, and Pseudomonas oryzihabitans) was associated with the spread of a skin lesion. The in vitro biofilm-forming capacity of strains isolated from the wound may represent the potential virulence of these strains. Thus, the role of polymicrobial biofilm may be crucial in ulcer formation and CTB manifestation. Conclusions: Severe wound healing as a unique biofilm-forming niche should be tested for Mycobacterium (on species and strain levels) and coexisting microorganisms using a wide range of microbiological techniques. In immunodeficient patients with non-typical CTB presentation, the chain of transmission and MTB spread is still an open issue for further research.

Cutaneous and Pulmonary Tuberculosis-Diagnostic and Therapeutic Difficulties in a Patient with Autoimmunity.

Cutaneous tuberculosis (CTB) is a very rare disease and accounts for only 1-2% of cases of extrapulmonary tuberculosis (EPTB). Due to the variety of its clinical manifestations, the uncharacteristic appearance of its lesions, resembling other dermatoses in the early stages, and the limited experience of clinicians due to the rarity of CTB, diagnosis is very difficult. Particularly noteworthy is the fact that most cases of EPTB, including skin tuberculosis (TB), can be a manifestation of systemic involvement. In this paper, we present a case of an immunocompromised patient who was diagnosed with CTB almost a year after the first dermatological lesions were located on the lower extremities. At the same time, due to respiratory symptoms, a diagnosis of pulmonary TB (PTB) was made, and radiological and microbiological confirmations were obtained.

Different Cutibacterium acnes Phylotypes Release Distinct Extracellular Vesicles.

Bacterial extracellular vesicles (EVs) perform various biological functions, including those that are critical to microbes. Determination of EVs composition allows for a deep understanding of their role in the bacterial community and communication among them. Cutibacterium acnes, formerly Propionibacteriumacnes, are commensal bacteria responsible for various infections, e.g., prosthesis, sarcoidosis, soft-tissue infections, and the most known but still controversial-acnes lesion. In C. acnes, three major phylotypes represented variable disease associations. Herein, for the first time, we present a comparative analysis of EVs obtained from three C. acnes phylotypes (IA1, IB, and II) to demonstrate the existence of differences in their protein and lipid composition. In the following work, the morphological analysis of EVs was performed, and the SDS-PAGE protein profile and the lipid profile were presented using the TLC and MALDI-TOF MS methods. This study allowed us to show major differences between the protein and lipid composition of C. acnes EVs. This is a clear indication that EVs released by different phylotypes of the one species are not identical to each other in terms of composition and should be separately analyzed each time to obtain reliable results.

Microbiome Analysis and Pharmacovigilance After Inhaled Glucocorticoid: Oral Dysbiosis With the Isolation of Three Rothia Species and Subsequent Sjögren's Syndrome.

Background: Treatment of respiratory tract diseases with inhaled glucocorticoids is a form of therapy that has been used for many years. It shows lower potency of side effects; nevertheless, microbiome change, sinopulmonary dysbiosis, secondary immunodeficiency, and immunomodulatory effects are underestimated. The latest guideline recommendations introduce the use of empirical antibiotic and/or multiplying inhaled glucocorticoids in therapeutic intervention of asthma and chronic pulmonary obstructive disease. Aims and objectives: The aim of the study was to describe a simple, universal, and cost-effective method of microbiome analysis for clinical trials. Such a general method for monitoring pharmacovigilance should be widely available and reliable. Methods: The study material included two kinds of swabs, taken from the same mouth ulcerations of patients with asthma treated with a temporary quadruple dose of fluticasone. The microbiological investigation was performed, and identification of the isolates was carried out using the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) Biotyper. Results: The analysis of dry swab demonstrated the presence of typical oral bacteria (Neisseria spp. and Streptococcus spp.), alongside with the potentially pathogenic Actinomyces spp. and three different Rothia species, identified simultaneously: R. aeria, R. dentocariosa, and R. mucilaginosa. Although quadrupled dose of corticoids was discontinued and ulcer healing was observed, the patients required topical therapy for maintained xerostomia. Progressive systemic autoimmunity (seronegative Sjögren's syndrome with major organ involvement) was observed later. Conclusion: Topical steroids (especially in quadruple dose) require attention to safety, immunomodulation, and microbiological outcome. They showed systemic side effects: microbiome alteration, humoral (IgG) immunodeficiency, and systemic autoimmunity. Isolation of three species of Rothia from a patient with mouth ulcers after steroid therapy suggests their participation in infectious and inflammatory processes. The proposed a methodology using MALDI-TOF-MS may be a prototype approach for microbial diagnostics in clinical trials of immunomodulatory drugs.

Timber-colonizing gram-negative bacteria as potential causative agents of respiratory diseases in woodworkers.

OCCURRENCE: Gram-negative bacteria occur commonly in the inner tissues of stored coniferous and deciduous timber, showing a marked variation in numbers. The greatest maximal numbers are found in the sapwood of coniferous timber. The common constituents of the Gram-negative biota are potentially pathogenic species of Enterobacteriaceae family of the genera Rahnella, Pantoea, Enterobacter, and Klebsiella. The air of wood-processing facilities is polluted with the wood-borne Gram-negative bacteria and produced by them endotoxin, as demonstrated worldwide by numerous studies. EFFECTS: There are three potential pathways of the pathogenic impact of wood-borne Gram-negative bacteria on exposed woodworkers: allergic, immunotoxic, and infectious. Allergic impact has been underestimated for a long time with relation to Gram-negative bacteria. Hopefully, the recent demonstration of the first documented case of hypersensitivity pneumonitis (HP) in woodworkers caused by Pantoea agglomerans which developed in extremely large quantities in birch sapwood, would speed up finding of new wood-related cases of HP caused by Gram-negative bacteria. The second pathway is associated with endotoxin, exerting strong immunotoxic (excessively immunostimulative) action. It has been demonstrated that endotoxin is released into wood dust in the form of nano-sized microvesicles, by peeling off the outer membrane of bacteria. Endotoxin microvesicles are easily inhaled by humans together with dust because of small dimensions and aerodynamic shape. Afterwards, they cause a nonspecific activation of lung macrophages, which release numerous inflammatory mediators causing an inflammatory lung reaction, chest tightness, fever, gas exchange disorders, and bronchospasm, without radiographic changes. The resulting disease is known as "Organic Dust Toxic Syndrome" or "toxic pneumonitis." The potential third pathway of pathogenic impact is infection. The suspected species is Klebsiella pneumoniae that may occur commonly in wood dust; however, until now this pathway has not been confirmed. CONCLUSION: Summarizing, Gram-negative bacteria-inhabiting timber should be considered, besides filamentous fungi and actinobacteria, as important risk factors of occupational disease in woodworkers that could be either HP with allergenic background or toxic pneumonitis elicited by endotoxin.

Lsr2 and Its Novel Paralogue Mediate the Adjustment of Mycobacterium smegmatis to Unfavorable Environmental Conditions.

Lsr2 is a nucleoid-associated protein (NAP) that has been found strictly in actinobacteria, including mycobacteria. It is a functional homolog of histone-like nucleoid-structuring protein (H-NS); it acts as a DNA-bridging protein that plays a role in chromosomal organization and transcriptional regulation. To date, the studies on Lsr2 have focused mainly on Mycobacterium tuberculosis In this study, we analyze the role of Lsr2 as a transcription factor in Mycobacterium smegmatis, a saprophytic bacterium whose natural habitat (soil and water) substantially differs from those of the obligatory mycobacterial pathogens. Our chromatin immunoprecipitation-sequencing (ChIP-seq) data revealed that Lsr2 binds preferentially to AT-rich regions of the M. smegmatis chromosome. We found that Lsr2 acts mainly as a repressor, controlling gene expression either directly by binding promoter regions or indirectly through DNA loop formation and DNA coating. One of the Lsr2-repressed genes encodes polyketide synthase (MSMEG_4727), which is involved in the synthesis of lipooligosaccharides (LOSs). An M. smegmatis strain deprived of Lsr2 produces more LOSs, which is mirrored by changes in the smoothness of cells and their susceptibilities to antibiotics. Unlike M. tuberculosis, M. smegmatis additionally encodes a paralogue of Lsr2, MSMEG_1060, which is a novel member of the mycobacterial NAP family. The Lsr2 and MSMEG_1060 proteins exhibit different DNA binding specificities and chromosomal localizations. Our results suggest that these proteins help M. smegmatis cells cope with stress conditions, including hypoxia and exposure to antibiotics. Thus, the present work provides novel insight into the role of Lsr2 paralogues in the ability of a saprophytic mycobacterial species to adjust to environmental changes.IMPORTANCE Nucleoid-associated proteins (NAPs) are the most abundant proteins involved in bacterial chromosome organization and global transcription regulation. The mycobacterial NAP family includes many diverse proteins; some are unique to actinobacteria, and many are crucial for survival under stress (e.g., HupB and Lsr2) and/or optimal growth conditions (e.g., mycobacterial integration host factor [mIHF]). Here, we present a comprehensive study concerning two functional homologues of mycobacterial H-NS: Lsr2 and its paralogue from M. smegmatis, MSMEG_1060. We found that Lsr2 plays a role in transcriptional regulation, mainly by repressing gene expression via DNA loop formation and/or DNA-coating mechanisms. Intriguingly, the number of Lsr2-mediated genes was found to increase under hypoxia. Compared to Lsr2, MSMEG_1060 exhibits a different DNA binding specificity and chromosomal localization. Since tuberculosis remains a serious worldwide health problem, studies on stress response-mediating agents, such as Lsr2, may contribute to the development of novel antituberculosis drugs.

Acute hypersensitivity pneumonitis in woodworkers caused by inhalation of birch dust contaminated with Pantoea agglomerans and Microbacterium barkeri.

CASE DESCRIPTION: Five workers (2 males and 3 females) employed in a furniture factory located in eastern Poland developed hypersensitivity pneumonitis (HP) after the pine wood used for furniture production was replaced by birch wood. All of them reported onset of respiratory and general symptoms (cough, shortness of breath, general malaise) after inhalation exposure to birch dust, showed crackles at auscultation, ground-glass attenuations in HRCT examination, and lymphocytosis in the BAL examination. The diagnosis of acute HP was set in 4 persons and the diagnosis of subacute HP in one. IDENTIFICATION OF SPECIFIC ALLERGEN: Samples of birch wood associated with evoking disease symptoms were subjected to microbiological analysis with the conventional and molecular methods. Two bacterial isolates were found to occur in large quantities (of the order 108 CFU/g) in examined samples: Gram-negative bacterium of the species Pantoea agglomerans and a non-filamentous Gram-positive actinobacterium of the species Microbacterium barkeri. In the test for inhibition of leukocyte migration, 4 out of 5 examined patients showed a positive reaction in the presence of P. agglomerans and 2 in the presence of M. barkeri. Only one person showed the presence of precipitins to P. agglomerans and none to M. barkeri. In the inhalation challenge, which is the most relevant allergological test in the HP diagnostics, all patients reacted positively to P. agglomerans and only one to M. barkeri. The results indicate that P. agglomerans developing in birch wood was the main agent causing HP in the workers exposed to the inhalation of dust from this wood, while the etiologic role of M. barkeri is probably secondary. CONCLUSION: The results demonstrate that apart from fungi and filamentous actinobacteria, regarded until recently as causative agents of HP in woodworkers, Gram-negative bacteria and non-filamentous actinobacteria may also elicit disease symptoms in the workers processing wood infected with large amounts of these microorganisms. The results obtained also seem to indicate that cellular-mediated reactions are more significant for causing disease symptoms compared to those that are precipitin-mediated.

Structural elucidation of Tsukamurella pulmonis neutral polysaccharide and its visualization in infected mouse tissues by specific monoclonal antibodies.

Tsukamurella pulmonis is an opportunistic actinomycetal pathogen associated with a variety of rarely diagnosed human infections. In clinical cases of infection, T. pulmonis usually accompanies other bacterial pathogens. Because of these mixed infections, a robust diagnostic assay is important. The bacteria cell surface polysaccharides are considered not only useful targets for diagnostics but also intriguing subjects for analysis of the interactions that regulate the host response in general. Here, the structure of the polysaccharide component of the T. pulmonis cell wall was established. Sugar and methylation analysis and 2D-NMR techniques revealed that its polysaccharide belongs to the class of arabinomannan composed of branched tetrasaccharide repeating units, with addition of linear →6)-α-D-Manp-(1→ mannan. Rabbit polyclonal sera against T. pulmonis and T. paurometabola bacterial cells revealed cross reactivity between their antigens. Tissue samples from mice infected with T. pulmonis revealed liver abscesses and pathologic granules located intracellularly when immunohistochemically stained with monoclonal antibodies raised against T. pulmonis polysaccharide. Ultrastructural studies revealed that these granules contain T. pulmonis cells. These observations indicate that T. pulmonis is a pathogenic species capable of spreading within the organism, presumably through the blood.

Elizabethkingia miricola as an opportunistic oral pathogen associated with superinfectious complications in humoral immunodeficiency: a case report.

BACKGROUND: Elizabethkingia miricola is a rare Gram-negative bacterium found in water and clinical specimens. Typical culturing methods often misidentify Elizabethkingia spp. as Flavobacterium or Chryseobacterium. Although diagnosis is based on culturing samples taken from sterile sites, such as blood, a proper identification of this bacterium requires an expertise that goes beyond the capabilities of a typical clinical laboratory. CASE PRESENTATION: A 35-year-old woman diagnosed with common variable immunodeficiency was admitted to our center. Previous treatment with antibiotics (amoxicillin plus clavulanate, first and third generation of cephalosporins, macrolides) and systemic corticosteroids (up to 120 mg/day of prednisolone) failed to arrest the spread of inflammation. Gingival recession was observed in her oral cavity, resulting in an apparent lengthening of her teeth. In addition to typical commensal bacteria, including streptococci and neisseriae, strains of Rothia mucilaginosa and Elizabethkingia miricola were identified upon a detailed microbiological examination using a MALDI-TOF MS Biotyper system. The presence of the latter strain correlated with severe periodontitis, lack of IgA in her saliva and serum, a very low IgG concentration (< 50 mg/dl), IgM-paraproteinemia, decreases in C3a and C5a and microvascular abnormality. High-dose immunoglobulin (to maintain IgG > 500 mg/dl) and targeted levofloxacin treatment resulted in immune system reconstitution, oral healing, and eradication of the Elizabethkingia infection. CONCLUSIONS: E. miricola rarely causes disease in healthy individuals. However, the overgrowth of commensal bacteria, lack of IgG/IgA, microvasculopathy and complement cascade activation in patients with humoral immunodeficiency may facilitate Elizabethkingia invasion. Overuse of antibiotics, particularly beta-lactams, may cause mucosal colonization by E. miricola, followed by its multiplication combined with periodontitis that prompts bacterial translocation. MALDI-TOF Biotyper analysis may become a method of choice for identification of Elizabethkingia infections.

CD1: A Singed Cat of the Three Antigen Presentation Systems.

Contrary to general view that the MHC Class I and II are the kapellmeisters of recognition and response to antigens, there is another big player in that part of immunity, represented by CD1 glycoproteins. In contrast to MHC Class I or II, which present peptides, CD1 molecules present lipids. Humans express five CD1 proteins (CD1a-e), four of which (CD1a-d) are trafficked to the cell surface, where they may display lipid antigens to T-cell receptors. This interaction may lead to both non-cognate and cognate T cell help to B cells, the latter eliciting anti-lipid antibody response. All CD1 proteins can bind a broad range of structurally different exogenous and endogenous lipids, but each shows a preference to one or more lipid classes. This unorthodox binding behavior is the result of elaborate architectures of CD1 binding clefts and distinct intracellular trafficking routes. Together, these features make CD1 system a versatile player in immune response, sitting at the crossroads of innate and adaptive immunity. While CD1 system may be involved in numerous infectious, inflammatory, and autoimmune diseases, its involvement may lead to opposite outcomes depending on different pathologies. Despite these ambiguities and complexity, CD1 system draws growing attention and continues to show glimmers of therapeutic potential. In this review, we summarize the current knowledge about CD1 proteins, their structures, lipid-binding profiles, and roles in immunity, and evaluate the role of CD1 proteins in eliciting humoral immune response.

The profile of polyunsaturated fatty acids in juvenile idiopathic arthritis and association with disease activity.

We investigated the association between dietary intake of n-3 and n-6 polyunsaturated fatty acids (PUFAs), serum profiles, and immune and inflammatory markers in juvenile idiopathic arthritis (JIA) in relation to onset, activity, and duration. A total of 66 JIA patients and 42 controls were included. Serum PUFA levels were assessed by gas-liquid chromatography-mass spectrometry, a dietary intake by 7-day dietary record method, and IL-6, IL-10, and IL-17A levels using ELISA. Dietary PUFA intake did not differ between the JIA group and controls. Intakes of n-6 and n-3 PUFA and serum levels were not associated. Levels of total n-6 PUFA and linoleic acid (LA) were higher in inactive JIA than in active JIA. Patients with active and short-lasting disease (less than 3 months from diagnosis) had significantly lower levels of arachidonic acid (AA) and docosahexaenoic acid (DHA) than the control. Serum α-linolenic acid (ALA) levels were significantly higher in poly-JIA than in oligo-JIA and in controls. We found significantly higher serum IL-10 levels in JIA than in controls. Serum n-6 and n-3 levels were significantly negatively correlated with active joint count, erythrocyte sedimentation rate, and C-reactive protein and positively with platelet count. Our study presents the low levels of AA and DHA in the active phase of short-lasting JIA, particularly poly-JIA, and the relationship between n-6 and n-3 PUFA and classic markers of inflammation. PUFAs may contribute to the pathogenesis of JIA and support a necessity to identify new targets suitable for successful interventional studies in JIA patients.

Lactobacillus johnsonii glycolipids, their structure and immunoreactivity with sera from inflammatory bowel disease patients.

Structural studies of the major glycolipids produced by two Lactobacillus johnsonii (LJ) strains, LJ 151 isolated from intestinal tract of healthy mice and LJ 142 isolated from mice with experimentally induced inflammatory bowel disease (IBD), were performed. Two major glycolipids, GL1 and GL2, were present in lipid extracts from L. johnsonii 142 and 151 strains. Glycolipid GL1 has been identified as ß-D-Glcp-(1→6)-α-D-Galp-(1→2)-α-D-Glcp-diglyceride and GL2 as α-D-Galp-(1→2)-α-D-Glcp-diglyceride. The main fatty acid residues identified by gas-liquid chromatography-mass spectrometry were palmitic, stearic and lactobacillic acids. Besides structural elucidation of the major glycolipids, the aim of this study was to determine the immunochemical properties of these glycolipids and to compare their immunoreactivity to that of polysaccharides obtained from the same strains. Sera from rabbits immunized with bacterial cells possessed much higher serological reactivity with polysaccharides than with glycolipids. Inversely, reactivity of the glycolipids with human sera from patients with IBD was much higher than that determined for the polysaccharides, while reactivity of glycolipids with human sera from healthy individuals was much lower than one measured for the polysaccharides. Results indicate that glycoconjugates from Lactobacillus cell wall act as antigens and may represent new IBD diagnostic biomarkers.

Isolation of Staphylococcus microti from milk of dairy cows with mastitis.

The present paper is a case-report of multiple udder infections in a dairy herd caused by Staphylococcus microti. Over a 22-month period, eleven S. microti isolates from milk samples from 9 cows were collected. The animals experienced subclinical (with one exception) intramammary infections with a high self-cure rate. The identification of the microorganism was carried out by means of two independent approaches: nucleotide sequence analysis of the 16S rRNA gene, as well as some housekeeping genes (sodA, rpoB, dnaJ), and matrix-assisted laser desorption ionization-time of flight mass spectrometry. All S. microti isolates belonged to an apparently single clone (as detected by the RAPD analysis), indicating that the microorganism could adapt, to some degree, to the bovine mammary gland or even spread from cow to cow in a contagious manner. This report is, to our knowledge, the first ever case of bovine mastitis caused by S. microti and the first instance of isolation of this microorganism from domesticated animals.

Creation of an In-House Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Corynebacterineae Database Overcomes Difficulties in Identification of Nocardia farcinica Clinical Isolates.

Nocardiosis is a rare disease that is caused by Gram-positive actinobacteria of the Nocardia genus and affects predominantly immunocompromised patients. In its disseminated form, it has a predilection for the central nervous system and is associated with high mortality rates. Therefore, prompt identification of the pathogen is critical. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry is a relatively novel technique used for identification of microorganisms. In this work, an upgraded MALDI-TOF Biotyper database containing Corynebacterineae representatives of strains deposited in the Polish Collection of Microorganisms was created and used for identification of the strain isolated from a nocardial brain abscess, mimicking a brain tumor, in an immunocompetent patient. Testing with the API Coryne system initially incorrectly identified Rhodococcus sp., while chemotaxonomic tests, especially mycolic acid analysis, enabled correct Nocardia identification only at the genus level. Subsequent sequence analysis of 16S rRNA and secA1 genes confirmed the identification. To improve the accuracy of the results, an in-house database was constructed using optimized parameters; with the use of the database, the strain was eventually identified as Nocardia farcinica. Clinical laboratories processing various clinical strains can upgrade a commercial database to improve and to accelerate the results obtained. This is especially important in the case of Nocardia, for which valid microbial diagnosis remains challenging; reference laboratories are often required to identify and to survey these rare actinobacteria.

An airborne actinobacteria Nocardiopsis alba isolated from bioaerosol of a mushroom compost facility.

Actinobacteria are widely distributed in many environments and represent the most important trigger to the occupant respiratory health. Health complaints, including hypersensitivity pneumonitis of the workers, were recorded in a mushroom compost facility (MCF). The studies on the airborne bacteria were carried out to find a possible microbiological source of these symptoms. Culture analysis of compost bioaerosols collected in different location of the MCF was performed. An assessment of the indoor microbial exposure revealed bacterial flora of bioaerosol in the mushroom compost facility represented by Bacillus, Geobacillus, Micrococcus, Pseudomonas, Staphylococcus spp., and actinobacterial strain with white aerial mycelium. The thermotolerant actinobacterial strain of the same morphology was repeatedly isolated from many locations in MCF: air, compost sample, and solid surface in production hall. On the base of complex morphological, chemotaxonomic, and phylogenetic characteristics, the isolate has been classified as Nocardiopsis alba. Dominant position of N. alba in microbial environment of the mushroom compost facility may represent an indicator microorganism in compost bioaerosol. The bioavailability of N. alba in mushroom compost facility creates potential risk for the health of workers, and the protection of respiratory tract and/or skin is strongly recommended.

The structure and immunoreactivity of exopolysaccharide isolated from Lactobacillus johnsonii strain 151.

The exopolysaccharide (EPS) structure from Lactobacillus johnsonii strain 151 isolated from the intestinal tract of mice was investigated. Sugar and methylation analyses together with (1)H and (13)C NMR spectroscopy, including two-dimensional (1)H,(1)H COSY, TOCSY, NOESY, and (1)H,(13)C HSQC, HMBC experiments, revealed that the repeating unit of the EPS is the linear pentasaccharide: →6)-α-d-Galp-(1→6)-α-d-Glcp-(1→3)-ß-d-Galf-(1→3)-α-d-Glcp-(1→2)-ß-d-Galf-(1→ The immunoreactivity of two structurally different exopolysaccharides isolated from L. johnsonii, 151 and 142 (Carbohydr. Res. 2010, 345, 108-114), was compared. Both EPSs differed in their reactivity with antisera. EPS from L. johnsonii 151 reacted with anti-Lactobacillus polyclonal sera against cells of five different strains, while EPS from L. johnsonii 142 was found to react only with its own antiserum. The broader specificity and higher reactivity of EPS from 151 strain than EPS from 142 strain were also observed with human sera. The physiological antibodies recognizing polysaccharide antigens were present in both adults and umbilical cord blood sera. A highly specific EPS 142 bearing strain was isolated from experimentally induced inflammatory bowel disease (IBD) mice, while a strain with EPS 151 isolated from the intestinal tract of healthy mice is characterized by a broad immune reactivity common structure.

[Identification of environmental Actinobacteria representing an occupational health risk]. / Identyfikacja aktynobakterii srodowiskowych stanowiacych potencjalne zagrozenie zawodowe.

Actinobacteria, the etiologic agents of tuberculosis, actinomycosis, respiratory infections and pathological skin lesions, are also classified as hazardous biological agents at the workplace. An increased number of Actinobacteria primarily occurs at the workplaces in composting plants, agriculture, waste management facilities, libraries and museums. Robust identification of Actinobacteria requires a polyphasic diagnostic strategy including an assessment of morphological, physiological, biochemical and chemotaxonomic features as well as genotyping. Commercially available diagnostic kits often do not include bacteria isolated from the environment and therefore analyses of chemotaxonomic markers--components of peptidoglycan, fatty acids, polar lipids (phospho- and glycolipids) and isoprenoid quinones are recommended. The paper discusses a comprehensive approach to the isolation and identification of Actinobacteria, with emphasis on chemotaxonomic methods. A diagnostic procedure is exemplified by environmental strains obtained from composting plants and libraries.

Immunochemical studies of trehalose-containing major glycolipid from Tsukamurella pulmonis.

The chemical structure of the major glycolipid present in the chloroform-methanol extract of bacterial biomass of Tsukamurella pulmonis is reported. This compound was purified by TLC and HPLC. The sugar analysis revealed only glucose. Detailed chemical analyses, NMR, and MALDI FT-ICR-mass spectrometric studies identified 2,3-di-O-acyl-alpha-d-glucopyranosyl-(1-->1)-alpha-d-glucopyranose as the final structure. Short branched fatty acids (4:0 or 5:0) were linked to C-3 and saturated, mono, and diunsaturated 18:0, 18:1, 18:2, 20:1, 20:2, and 20:0 to C-2 of the same glucose residue. ELISA tests revealed the weak cross-reactivity of the glycolipid with rabbit antisera against cells of T. paurometabola, Rhodococcus wratislaviensis, and Nocardiopsis dassonvillei.