Intrathecal antibody responses to GalC in Guillain-Barré syndrome triggered by Mycoplasma pneumoniae.

Mycoplasma pneumoniae (Mp) triggers Guillain-Barré syndrome (GBS) and elicits anti-galactocerebroside (GalC) antibodies. Specifically anti-GalC IgG is associated with Mp-GBS, possibly because of its better ability to cross the blood-nerve barrier (BNB). We here investigated CSF for the presence of anti-GalC in GBS. Intrathecal anti-GalC was found in 46% of Mp-GBS patients (n=6/13), in contrast to 16% of GBS controls (n=4/25) and 0% of non-GBS controls (n=0/7). The antibodies most likely originated from increased BNB permeability and/or intrathecal synthesis. Intrathecal anti-GalC IgG was specifically associated with Mp-GBS, further supporting that anti-GalC IgG contributes to the pathogenesis of GBS.

Progressive Dispersion of Azole Resistance in Aspergillus fumigatus: Fatal Invasive Aspergillosis in a Patient with Acute Myeloid Leukemia Infected with an A. fumigatus Strain with a cyp51A TR46 Y121F M172I T289A Allele.

Patients with hematologic malignancies as well as allogeneic hematopoietic stem cell transplantation (HSCT) patients are at high risk for invasive aspergillosis. Here, we report a culture- and autopsy-proven fatal invasive aspergillosis in an allogeneic HSTC patient which he developed despite posaconazole prophylaxis. The agent was determined to be an azole-resistant Aspergillus fumigatus strain bearing the cyp51A mutation combination TR46 Y121F M172I T289A. At increasing frequency, the azole resistance of A. fumigatus is being reported globally, limiting treatment options and complicating regimens.

First report on sepsis caused by Porphyromonas pogonae.

We report on a 62 year old patient who developed sepsis due to an infection caused by Porphyromonas pogonae, a recently described species of the bacterial genus Porphyromonas. This is the first case of an invasive infection with this pathogen.

The surface-displayed chaperones GroEL and DnaK of Mycoplasma pneumoniae interact with human plasminogen and components of the extracellular matrix.

Mycoplasma pneumoniae is a common cause of community-acquired infections of the human respiratory tract. The strongly reduced genome of the cell wall-less bacteria results in limited metabolic pathways and a small number of known virulence factors. In addition to the well-characterized adhesion apparatus and the expression of tissue-damaging substances, surface-exposed proteins with a primary function in cytosol-located processes such as glycolysis have been attracting attention in recent years. Due to interactions with host factors, it has been suggested that these bacterial proteins contribute to pathogenesis. Here, we investigated the chaperones GroEL and DnaK of M. pneumoniae as candidates for such moonlighting proteins. After successful expression in Escherichia coli and production of polyclonal antisera, the localization of both chaperones on the surface of bacteria was confirmed. Binding of recombinant GroEL and DnaK to human A549 cells, to plasminogen as well as to vitronectin, fibronectin, fibrinogen, lactoferrin and laminin was demonstrated. In the presence of both recombinant proteins and host activators, plasminogen can be activated to the protease plasmin, which is able to degrade vitronectin and fibrinogen. The results of the study extend the spectrum of surface-exposed proteins in M. pneumoniae and indicate an additional role of both chaperones in infection processes.

Interactions of surface-displayed glycolytic enzymes of Mycoplasma pneumoniae with components of the human extracellular matrix.

Mycoplasma pneumoniae is a major cause of community-acquired respiratory infections worldwide. Due to the strongly reduced genome, the number of virulence factors expressed by this cell wall-less pathogen is limited. To further understand the processes during host colonization, we investigated the interactions of the previously confirmed surface-located glycolytic enzymes of M. pneumoniae (pyruvate dehydrogenase A-C [PdhA-C], glyceraldehyde-3-phosphate dehydrogenase [GapA], lactate dehydrogenase [Ldh], phosphoglycerate mutase [Pgm], pyruvate kinase [Pyk] and transketolase [Tkt]) to the human extracellular matrix (ECM) proteins fibrinogen (Fn), fibronectin (Fc), lactoferrin (Lf), laminin (Ln) and vitronectin (Vc), respectively. Concentration-dependent interactions between Fn and Vc and all eight recombinant proteins derived from glycolytic enzymes, between Ln and PdhB-C, GapA, Ldh, Pgm, Pyk and Tkt, between Lf and PdhA-C, GapA and Pyk, and between Fc and PdhC and GapA were demonstrated. In most cases, these associations are significantly influenced by ionic forces and by polyclonal sera against recombinant proteins. In immunoblotting, the complex of human plasminogen, activator (tissue-type or urokinase plasminogen activator) and glycolytic enzyme was not able to degrade Fc, Lf and Ln, respectively. In contrast, degradation of Vc was confirmed in the presence of all eight enzymes tested. Our data suggest that the multifaceted associations of surface-localized glycolytic enzymes play a potential role in the adhesion and invasion processes during infection of human respiratory mucosa by M. pneumoniae.

Mycoplasma pneumoniae triggering the Guillain-Barré syndrome: A case-control study.

OBJECTIVE: Guillain-Barré syndrome (GBS) is an acute postinfectious immune-mediated polyneuropathy. Although preceding respiratory tract infections with Mycoplasma pneumoniae have been reported in some cases, the role of M. pneumoniae in the pathogenesis of GBS remains unclear. We here cultured, for the first time, M. pneumoniae from a GBS patient with antibodies against galactocerebroside (GalC), which cross-reacted with the isolate. This case prompted us to unravel the role of M. pneumoniae in GBS in a case-control study. METHODS: We included 189 adults and 24 children with GBS and compared them to control cohorts for analysis of serum antibodies against M. pneumoniae (n = 479) and GalC (n = 198). RESULTS: Anti-M. pneumoniae immunoglobulin (Ig) M antibodies were detected in GBS patients and healthy controls in 3% and 0% of adults (p = 0.16) and 21% and 7% of children (p = 0.03), respectively. Anti-GalC antibodies (IgM and/or IgG) were found in 4% of adults and 25% of children with GBS (p = 0.001). Anti-GalC-positive patients showed more-frequent preceding respiratory symptoms, cranial nerve involvement, and a better outcome. Anti-GalC antibodies correlated with anti-M. pneumoniae antibodies (p < 0.001) and cross-reacted with different M. pneumoniae strains. Anti-GalC IgM antibodies were not only found in GBS patients with M. pneumoniae infection, but also in patients without neurological disease (8% vs 9%; p = 0.87), whereas anti-GalC IgG was exclusively found in patients with GBS (9% vs 0%; p = 0.006). INTERPRETATION: M. pneumoniae infection is associated with GBS, more frequently in children than adults, and elicits anti-GalC antibodies, of which specifically anti-GalC IgG may contribute to the pathogenesis of GBS. Ann Neurol 2016;80:566-580.

Emergence of Mycoplasma genitalium strains showing mutations associated with macrolide and fluoroquinolone resistance in the region Dresden, Germany.

Among 323 specimens from male patients with symptoms of non-gonococcal urethritis, Mycoplasma genitalium was detected in 19 samples by real-time PCR. Mutations of 23S rRNA gene associated with macrolide resistance were confirmed in 10 strains. Amino acid changes at positions 81 and 83 of ParC protein were demonstrated indicating quinolone resistance of two strains.

Antibody Response to Mycoplasma pneumoniae: Protection of Host and Influence on Outbreaks?

In humans of all ages, the cell wall-less and genome-reduced species Mycoplasma pneumoniae can cause infections of the upper and lower respiratory tract. The well-documented occurrence of major peaks in the incidence of community-acquired pneumonia cases reported world-wide, the multifaceted clinical manifestations of infection and the increasing number of resistant strains provide reasons for ongoing interest in the pathogenesis of mycoplasmal disease. The results of recent studies have provided insights into the interaction of the limited virulence factors of the bacterium with its host. In addition, the availability of complete M. pneumoniae genomes from patient isolates and the development of proteomic methods for investigation of mycoplasmas have not only allowed characterization of sequence divergences between strains but have also shown the importance of proteins and protein parts for induction of the immune reaction after infection. This review focuses on selected aspects of the humoral host immune response as a factor that might influence the clinical course of infections, subsequent protection in cases of re-infections and changes of epidemiological pattern of infections. The characterization of antibodies directed to defined antigens and approaches to promote their induction in the respiratory mucosa are also preconditions for the development of a vaccine to protect risk populations from severe disease due to M. pneumoniae.

Network of Surface-Displayed Glycolytic Enzymes in Mycoplasma pneumoniae and Their Interactions with Human Plasminogen.

In different bacteria, primarily cytosolic and metabolic proteins are characterized as surface localized and interacting with different host factors. These moonlighting proteins include glycolytic enzymes, and it has been hypothesized that they influence the virulence of pathogenic species. The presence of surface-displayed glycolytic enzymes and their interaction with human plasminogen as an important host factor were investigated in the genome-reduced and cell wall-less microorganism Mycoplasma pneumoniae, a common agent of respiratory tract infections of humans. After successful expression of 19 glycolytic enzymes and production of polyclonal antisera, the localization of proteins in the mycoplasma cell was characterized using fractionation of total proteins, colony blot, mild proteolysis and immunofluorescence of M. pneumoniae cells. Eight glycolytic enzymes, pyruvate dehydrogenases A to C (PdhA-C), glyceraldehyde-3-phosphate dehydrogenase (GapA), lactate dehydrogenase (Ldh), phosphoglycerate mutase (Pgm), pyruvate kinase (Pyk), and transketolase (Tkt), were confirmed as surface expressed and all are able to interact with plasminogen. Plasminogen bound to recombinant proteins PdhB, GapA, and Pyk was converted to plasmin in the presence of urokinase plasminogen activator and plasmin-specific substrate d-valyl-leucyl-lysine-p-nitroanilide dihydrochloride. Furthermore, human fibrinogen was degraded by the complex of plasminogen and recombinant protein PdhB or Pgm. In addition, surface-displayed proteins (except PdhC) bind to human lung epithelial cells, and the interaction was reduced significantly by preincubation of cells with antiplasminogen. Our results suggest that plasminogen binding and activation by different surface-localized glycolytic enzymes of M. pneumoniae may play a role in successful and long-term colonization of the human respiratory tract.

New insights in the outbreak pattern of Mycoplasma pneumoniae.

Since a well-documented incidence peak in 2011/12 in European countries, infections due to the cell wall-less bacterium Mycoplasma pneumoniae have gained the increased attention of clinicians, microbiologists and health authorities. Despite the mild or asymptomatic clinical course of most M. pneumoniae infections, the microorganism is responsible for severe interstitial pneumonia and extra-pulmonary complications. Here, we report the time-dependence of 5545 notified cases of laboratory-confirmed M. pneumoniae disease in Saxony from 2001 until June 2014 as measured by serodiagnosis. In parallel, from 2003 until 2012 467 M. pneumoniae-positive respiratory samples or isolated strains were analysed by molecular typing based on sequence differences in the main P1 adhesin of M. pneumoniae. The epidemiological data showed a prolonged outbreak especially in the period 2011-2013. The typing of circulating strains during the outbreak did not support predominance of one of the two major P1 subtypes (mean proportion of subtype 1: 57%) or a change of one to the other subtype during the endemic situation before and during the outbreak period. From the last major outbreak in Europe, we conclude that the notification of M. pneumoniae-positive cases, which is legally required only in Saxony, should be expanded to the whole country, to optimise awareness of this human pathogen and to reflect upon antibiotic therapy.

Severe childhood Guillain-Barré syndrome associated with Mycoplasma pneumoniae infection: a case series.

We report seven children with recent Mycoplasma pneumoniae infection and severe Guillain-Barré syndrome (GBS) that presented to two European medical centres from 1992 to 2012. Severe GBS was defined as the occurrence of respiratory failure, central nervous system (CNS) involvement, or death. Five children had GBS, one Bickerstaff brain stem encephalitis (BBE), and one acute-onset chronic inflammatory demyelinating polyneuropathy (A-CIDP). The five patients with severe GBS were derived from an original cohort of 66 children with GBS. In this cohort, 17 children (26%) had a severe form of GBS and 47% of patients with M. pneumoniae infection presented with severe GBS. Of the seven patients in this case series, five were mechanically ventilated and four had CNS involvement (two were comatose). Most patients presented with non-specific clinical symptoms (nuchal rigidity and ataxia) and showed a rapidly progressive disease course (71%). Antibodies against M. pneumoniae were detected in all patients and were found to be intrathecally synthesised in two cases (GBS and BBE), which proves intrathecal infection. One patient died and only two patients recovered completely. These cases illustrate that M. pneumoniae infection in children can be followed by severe and complicated forms of GBS. Non-specific clinical features of GBS in such patients may predispose a potentially life-threatening delay in diagnosis.

Subunits of the Pyruvate Dehydrogenase Cluster of Mycoplasma pneumoniae Are Surface-Displayed Proteins that Bind and Activate Human Plasminogen.

The dual role of glycolytic enzymes in cytosol-located metabolic processes and in cell surface-mediated functions with an influence on virulence is described for various micro-organisms. Cell wall-less bacteria of the class Mollicutes including the common human pathogen Mycoplasma pneumoniae possess a reduced genome limiting the repertoire of virulence factors and metabolic pathways. After the initial contact of bacteria with cells of the respiratory epithelium via a specialized complex of adhesins and release of cell-damaging factors, surface-displayed glycolytic enzymes may facilitate the further interaction between host and microbe. In this study, we described detection of the four subunits of pyruvate dehydrogenase complex (PDHA-D) among the cytosolic and membrane-associated proteins of M. pneumoniae. Subunits of PDH were cloned, expressed and purified to produce specific polyclonal guinea pig antisera. Using colony blotting, fractionation of total proteins and immunofluorescence experiments, the surface localization of PDHA-C was demonstrated. All recombinant PDH subunits are able to bind to HeLa cells and human plasminogen. These interactions can be specifically blocked by the corresponding polyclonal antisera. In addition, an influence of ionic interactions on PDHC-binding to plasminogen as well as of lysine residues on the association of PDHA-D with plasminogen was confirmed. The PDHB subunit was shown to activate plasminogen and the PDHB-plasminogen complex induces degradation of human fibrinogen. Hence, our data indicate that the surface-associated PDH subunits might play a role in the pathogenesis of M. pneumoniae infections by interaction with human plasminogen.

Mycoplasma pneumoniae and Chlamydia spp. infection in community-acquired pneumonia, Germany, 2011-2012.

Mycoplasma pneumoniae and Chlamydia spp., which are associated with community-acquired pneumonia (CAP), are difficult to propagate, and can cause clinically indistinguishable disease patterns. During 2011-2012, we used molecular methods to test adult patients in Germany with confirmed CAP for infection with these 2 pathogens. Overall, 12.3% (96/783) of samples were positive for M. pneumoniae and 3.9% (31/794) were positive for Chlamydia spp.; C. psittaci (2.1%) was detected more frequently than C. pneumoniae (1.4%). M. pneumoniae P1 type 1 predominated, and levels of macrolide resistance were low (3.1%). Quarterly rates of M. pneumoniae-positive samples ranged from 1.5% to 27.3%, showing a strong epidemic peak for these infections, but of Chlamydia spp. detection was consistent throughout the year. M. pneumoniae-positive patients were younger and more frequently female, had fewer co-occurring conditions, and experienced milder disease than did patients who tested negative. Clinicians should be aware of the epidemiology of these pathogens in CAP.

Molecular characterization of macrolide resistance of a Mycoplasma pneumoniae strain that developed during therapy of a patient with pneumonia.

The development of macrolide resistance that occurred during 3 days of therapy with azithromycin to treat Mycoplasma pneumoniae pneumonia in a paediatric patient is reported. After extended molecular characterization of strains, the parallel occurrence of clones showing the non-mutated wild-type 23S rRNA sequence as well as mutations A2063G and A2064G, which are both responsible for phenotypic resistance, was confirmed for the first time.

Evaluation of five real-time PCR assays for detection of Mycoplasma pneumoniae.

Four commercial real-time PCR assays to detect Mycoplasma pneumoniae were tested, and the results were compared with the results for an in-house approach. Despite differences of crossing threshold values of up to 4, assays were able to detect at least 20 CFU/5 µl (52 fg DNA/5 µl) of sample with the Diagenode kit showing the best clinical sensitivity.

Preparation of melt-spun antimicrobially modified LDH/polyolefin nanocomposite fibers.

Layered double hydroxide (LDH) was synthesized and organically modified with camphorsulfonic acid (CSA) and ciprofloxacin. The thermal stability of CSA was improved remarkably under LDH shielding. A minimal inhibitory concentration of free CSA against tested bacteria was determined in order to define the essential quantity in LDH modification. The modified LDHs were melt-compounded with high density polyethylene and the prepared nanocomposites were further melt-spun using a piston-type spinning device. The melt-spun fibers were tested for their antimicrobial activity against Escherichia coli, Proteus vulgaris, Pseudomonas aeruginosa, Enterobacter cloacae, Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus pyogenes. CSA integrated fibers show susceptibility against Gram-positive bacteria and ciprofloxacin integrated fibers showed activity against both Gram-positive and Gram-negative bacteria.

Mycoplasma pneumoniae intrathecal antibody responses in Bickerstaff brain stem encephalitis.

The pathogenesis of Mycoplasma pneumoniae encephalitis is not established. We report, for the first time, the case of a patient with severe Bickerstaff brain stem encephalitis in whom we detected intrathecal production of M. pneumoniae-specific antibodies, contrasting the findings in another patient with less severe encephalitis in whom we detected intrathecal M. pneumoniae DNA but no specific antibodies. Our observations suggest that intrathecal M. pneumoniae-specific antibody responses may contribute to a more severe course of M. pneumoniae encephalitis.

Actinobacillus equuli ssp. haemolyticus in a semi-occlusively treated horse bite wound in a 2-year-old girl.

We report on the isolation of Actinobacillus equuli ssp. haemolyticus from wound smears of a 2-year-old girl who was admitted to the hospital due to partial amputation of the distal phalanx of her right middle finger caused by a horse bite. A. equuli typically causes diseases in horses and only very few reports describing human infections (mostly associated with wounds) are available in the literature. Interestingly, although the bacteria could be found in consecutive samples taken at different points in time, there were no signs of advancing infection or inflammation. Moreover, the fingertip regenerated after 74 days under semi-occlusive dressings with very pleasant results. For strain identification two automated systems were employed producing discrepant results: VITEK 2 described the pathogens as Pasteurella pneumotropica while MALDI-TOF MS analysis revealed A. equuli. Sequence analysis of 16S rDNA gene finally confirmed A. equuli ssp. haemolyticus as the isolated strain. The antimicrobial susceptibility testing was performed according to the CLSI criteria for Pasteurella spp. Additionally we conducted a test according to the EUCAST criteria.

Development of protective anti-Mycoplasma pneumoniae antibodies after immunization of guinea pigs with the combination of a P1-P30 chimeric recombinant protein and chitosan.

The attachment organelle of the human respiratory tract pathogen Mycoplasma pneumoniae is essential for colonization of the host mucosa. Furthermore, adherence-related proteins such as the major adhesin P1 and protein P30 represent vaccine candidates. Using the chimeric recombinant protein HP14/30, which combines surface-localized and adherence-involved regions of both proteins, we developed an optimized strategy to immunize guinea pigs. The vaccination protocol includes subcutaneous prime immunization followed by presentation of the antigen directly to the respiratory mucosa by two intranasal (i.n.) administrations and combination of antigen with the mucosal adjuvant chitosan. The immunization scheme induced high, consistent and long-lasting IgA levels in respiratory tract samples (BAL, nasal and throat washing fluid) from the animals. In comparison with a preimmune serum, incubation of M. pneumoniae cells with sera from these animals reduced the mean adhesion of bacteria to HeLa cells to 6%. After i.n. infection, immunized animals showed significantly decreased numbers of M. pneumoniae-specific genome copies, especially in the upper respiratory tract, in comparison with the control group. The results demonstrated that optimized immunization with the chimeric protein HP14/30 is promising for further vaccination efforts to prevent host colonization with M. pneumoniae.