Analysis of Risk Factors of Bronchiolitis Obliterans in Children with Mycoplasma pneumoniae Bronchiolitis.

OBJECTIVE: To investigate the related risk factors for bronchiolitis obliterans (BO) in children with mycoplasma pneumonia (MP) bronchiolitis. METHOD: The clinical data of 227 children with MP bronchiolitis who were admitted to the II Department of Respiratory of Children's Hospital of Hebei Province from January 2018 to June 2020 were retrospectively analyzed. According to the sequelae of BO, they were divided into 32 cases in the BO group and 195 cases in the non-BO group. The univariate analysis was performed on the clinical and laboratory parameters of the two groups, and the multifactor logistic regression was performed further to determine the independent risk factors for the occurrence of BO in MP bronchiolitis, and then, the cut-off value with the maximum diagnostic value of indicators was found through the ROC curve analysis. RESULTS: The results of univariate and multivariate logistic regression analysis showed that the independent risk factors for the occurrence of BO in MP bronchioles were longer duration of moist rales (OR = 1.203, P = 0.003), higher levels of serum lactate dehydrogenase (LDH) (OR = 1.005, P = 0.036), hypoxemia (OR = 7.442, P = 0.035), and pleural effusion (OR = 4.437, P = 0.004). The area under the ROC curve was 78.2%, 72.0%, 68.2%, and 71.0%, respectively (P < 0.001). The cut-off value of duration of moist rales and levels of serum LDH are 7.5 d and 330 U/L, respectively. CONCLUSION: Children with MP bronchiolitis with high serum LDH level (≥330 U/L), combined with hypoxemia, pleural effusion, and lung wet rale duration (≥7.5 d), may be more prone to BO, in which lung wet rale duration prediction value is the largest. Among them, duration of pulmonary moist rales has the highest predictive value.

Prevalence and molecular detection of contagious bovine pleuropneumonia in large ruminants in Punjab, Pakistan.

Contagious bovine pleuropneumonia (CBPP) is a highly contagious disease of cattle caused by Mycoplasma mycoides subsp. mycoides. It is characterized by anorexia, fever, dyspnea, polypnea, cough, and nasal discharges. Gross lesions in the lung such as marbling, sequestra, thickening of interlobular septa, and consolidation are evident. Serological tests including complement fixation test and competitive enzyme-linked immunosorbent assay and molecular tests such as polymerase chain reactions are used for diagnostic purposes. In this study, lung samples of suspected large ruminants (cattle n=560, buffalo n=293) were collected from abattoirs of three districts of Punjab namely Lahore, Kasur and Jhang. PCR was performed with specific primers, targeting the 16S ribosomal RNA gene to detect the positive cases. The results indicated that 49 samples (8.75%) of cattle were positive, with maximum prevalence was observed in Jhang with 16 positive samples (10.06%), but CBPP was not detected in any buffalo sample. High prevalence of disease was seen in cattle of more than seven years of age, in female cattle, and in cross-bred cattle. Age and gender were found significantly associated (P<0.05) with the prevalence of the disease. Gene sequencing of identified 5 isolates of Mycoplasma mycoides subsp. mycoides had more than 99% similarities with the strains isolated from China, Italy, Australia and Tanzania and were categorized into a monophyletic group but strain isolated from Portugal had more than 55% variable regions, hence clustered separately. This study confirms the presence of contagious bovine pleuropneumonia in the country which can be a threat to the livestock export market and warrants the implementation of control measures to mitigate the economic losses associated with the disease.

The Mycoplasma pneumoniae HapE alters the cytokine profile and growth of human bronchial epithelial cells.

Mycoplasma pneumoniae is one of the most common pathogenic causes of community-acquired pneumonia. Hydrogen sulfide, alanine, and pyruvate producing enzyme (HapE) is a recently discovered M. pneumoniae virulence factor that can produce H2S to promote erythrocyte lysis. However, other cytotoxic effects of HapE have not been explored. The present study examined the effects of this enzyme on normal human bronchial epithelial (NHBE) cells, in an attempt to identify additional mechanisms of M. pneumoniae pathogenesis. Recombinant HapE was purified for use in downstream assays. MTT and colony formation assays were conducted to determine the effects of HapE on cell viability and growth, while flow cytometry was used to examine changes in cell proliferation and cell cycle function. ELISA was performed to examine changes in the cytokine profile of HapE-treated cells. HapE treatment arrested NHBE cells in S phase and inhibited cell proliferation in a concentration-dependent manner. The anti-inflammatory factors interleukin (IL)-4 and IL-6 were significantly enhanced following HapE treatment. Increased secretion of pro-inflammatory factors was not observed. The effects of HapE on the respiratory epithelium may have an impact on the efficiency of host immune surveillance and pathogen elimination, and contribute to the pathogenesis of M. pneumoniae.

Serum lactate dehydrogenase isoenzymes 4 plus 5 is a better biomarker than total lactate dehydrogenase for refractory Mycoplasma pneumoniae pneumonia in children.

Although usually self-limiting, Mycoplasma pneumoniae pneumonia (MPP) may lead to clinical or radiological deterioration despite macrolide antibiotic therapy, resulting in the development of refractory MPP (RMPP). Corticosteroids have been used to treat RMPP with beneficial effects. Serum lactate dehydrogenase (LDH) is a suggested biomarker for the use of steroid therapy. Since serum LDH is a non-specific marker and elevated in many inflammatory processes, this study investigates the predicting level of LDH isoenzymes for RMPP. Fifty-four children with non-refractory M. pneumoniae pneumonia (NRMPP) and 16 children with RMPP were enrolled in this study. In comparison to the NRMPP group, the RMPP group showed significantly higher levels of serum LDH. Concerning LDH isoenzymes, the RMPP group showed significantly lower proportions of LDH1 and LDH2, while higher LDH4 and LDH5 percentage. Receiver operating characteristic curve analysis showed that the area under the curve for the total LDH data was 0.812 with a cut-off of 408 IU/L (sensitivity of 75.0%, specificity of 72.2%). The areas under the curve for LDH4, LDH5, and [LDH4 + LDH5] were estimated to be 0.813, 0.818, and 0.829, respectively. The threshold for [LDH4 + LDH5] was estimated to be 109.4 IU/L (sensitivity, 75.0%; specificity, 87.0%). These results indicate that for the initiation of corticosteroid therapy, serum [LDH4 + LDH5] level is a more sensitive biomarker than total LDH.

Pattern and Significance of Asymptomatic Elevation of Liver Enzymes in Mycoplasma Pneumonia in Children.

Mycoplasma infection is on the rise in recent times. It usually infects any system, including liver. This study aims to show the significance of elevated liver enzymes in mycoplasma pneumonia and to have a look at future prospects. This is a single-center retrospective study involving 105 children serologically positive for mycoplasma IgM and IgG antibodies and 50 with community-acquired pneumonia caused by organisms other than mycoplasma and Epstein-Barr virus from June 2015 to June 2016 and all without prior liver disease. The patients were followed after 10 days (7-14 days). The liver enzymes were significantly elevated in Mycoplasma pneumoniae infection. The mean levels of alanine transaminase and aspartate transaminase were 39.3 and 32.5 IU/L, respectively. There was a seasonal variation during the months of September and February. Liver involvement in mycoplasma pneumonia is mostly a benign condition and asymptomatic. It is insisted that children with continued elevation should be followed conservatively to avoid unnecessary diagnostic procedures in the future.

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.

Correlation of bacterial coinfection versus matrix metalloproteinase 9 and tissue inhibitor of metalloproteinase 1 expression in aortic aneurysm and atherosclerosis.

BACKGROUND: We searched for any relationship between Chlamydophila pneumoniae, Mycoplasma pneumoniae, matrix metalloproteinase 9 (MMP-9), and tissue inhibitor of metalloproteinase 1 (TIMP-1) in aneurysmatic atherosclerotic lesions, and whether this relationship differed from that in atherosclerotic nonaneurysmatic lesions. METHODS: Twenty-eight tissue samples paired by age and sex were grouped as follows: group 1 included 14 nonaneurysmal atherosclerotic fragments obtained from abdominal aortas collected from necropsies; group 2 included 14 aneurysmatic atherosclerotic aortic fragments obtained from patients during corrective surgery. Immunohistochemistry reactions were evaluated for C pneumoniae, M pneumoniae, MMP-9, and TIMP-1 antigens. Both groups were compared using the Mann-Whitney test, and the correlations among variables were obtained using the Spearman correlation test. P ≤ 0.05 was considered statistically significant. RESULTS: C pneumoniae and M pneumoniae antigens were detected in 100% of cases. A higher amount of C pneumoniae (P = 0.005), M pneumoniae (P = 0.002), and MMP-9 (P = 0.021) was found in adventitia of group 2 with aneurysm. A positive correlation was found in the aneurysm group, as follows: intima C pneumoniae versus adventitia thickness (r = 0.70; P = 0.01), media C pneumoniae versus adventitia C pneumoniae (r = 0.75; P = 0.002), intima C pneumoniae versus media C pneumoniae (r = 0.8; P = 0.00), and adventitia C pneumoniae versus intima M pneumoniae (r = 0.54; P = 0.05); negative correlations were as follows: adventitia thickness and adventitia M pneumoniae (r = -0.65; P = 0.01), media MMP-9 and media thickness (r = -0.55; P = 0.04), TIMP-1 media versus adventitia C pneumoniae (r = -0.86; P = 0.00), and TIMP-1 media versus M pneumoniae intima (r = -0.67; P = 0.03). Nonaneurysmal atherosclerotic group 1 results are as follows: adventitia C pneumoniae versus TIMP-1 media (r = 0.75; P = 0.01) and media C pneumoniae and adventitia C pneumoniae (r = 0.59; P = 0.03). CONCLUSIONS: The present work favors a role for coinfection of both M pneumoniae and C pneumoniae in the development of aortic atherosclerotic aneurysm, with increased adventitial inflammation, inhibition of TIMP-1 activity, and increased collagen degradation.

Mycoplasma pneumoniae in adult community-acquired pneumonia increases matrix metalloproteinase-9 serum level and induces its gene expression in peripheral blood mononuclear cells.

BACKGROUND: The objective of this study was to assess the concentration of metalloproteinase-2 (MMP-2) and metalloproteinase-9 (MMP-9) in peripheral circulation and their mRNA expression in peripheral blood mononuclear cells (PBMCs) in patients with CAP caused by M. pneumoniae. MATERIAL/METHODS: We prospectively analyzed MMPs in 40 hospitalized patients with M. pneumoniae CAP on admission, and in the convalescent phase. Twenty healthy men were used as controls. Quantitative real-time PCR and ELISA tests were used. RESULTS: MMP-9 mRNA expression in PBMCs was increased in the acute phase of illness compared to the control group as well as in convalescent phase in which case it was statistically significant (Mann-Whitney; p=0.028). The same was found for MMP-9 plasma levels (Mann-Whitney test; p<0.001; p=0.001). Circulating MMP-2 concentration in acute patients was significantly lower than in the control group and convalescent phase (Mann-Whitney test; p=0.012; p=0.001), while no MMP-2 mRNA expression was found in PBMCs. The plasma level of MMP-9 correlated with leukocyte count in peripheral circulation (r=0.67, p<0.001). CONCLUSIONS: We conclude that M. pneumoniae in adult CAP induces activity of MMP-9 in peripheral blood circulation.

Role of RecJ-like protein with 5'-3' exonuclease activity in oligo(deoxy)nucleotide degradation.

RecJ-like proteins belonging to the DHH family have been proposed to function as oligoribonucleases and 3'-phosphoadenosine 5'-phosphate (pAp) phosphatases in bacteria and archaea, which do not have Orn (oligoribonuclease) and CysQ (pAp phosphatase) homologs. In this study, we analyzed the biochemical and physiological characterization of the RecJ-like protein TTHA0118 from Thermus thermophilus HB8. TTHA0118 had high enzymatic activity as an oligodeoxyribonucleotide- and oligoribonucleotide-specific exonuclease and as pAp phosphatase. The polarity of degradation was 5' to 3', in contrast to previous reports about Bacillus subtilis NrnA, a RecJ-like protein. TTHA0118 preferentially hydrolyzed short oligodeoxyribonucleotides and oligoribonucleotides, whereas the RecJ exonuclease from T. thermophilus HB8 showed no such length dependence on oligodeoxyribonucleotide substrates. An insertion mutation of the ttha0118 gene led to growth reduction in minimum essential medium. Added 5'-mononucleotides, nucleosides, and cysteine increased growth of the ttha0118 mutant in minimum essential medium. The RecJ-like protein Mpn140 from Mycoplasma pneumoniae M129, which cannot synthesize nucleic acid precursors de novo, showed similar biochemical features to TTHA0118. Furthermore, B. subtilis NrnA also hydrolyzed oligo(deoxy)ribonucleotides in a 5'-3' direction. These results suggested that these RecJ-like proteins act in recycling short oligonucleotides to mononucleotides and in controlling pAp concentrations in vivo.

Inhibition of 5,10-methenyltetrahydrofolate synthetase.

The interaction of 5-formyltetrahydrofolate analogs with murine methenyltetrahydrofolate synthetase (MTHFS) was investigated using steady-state kinetics, molecular modeling, and site-directed mutagenesis. MTHFS catalyzes the irreversible cyclization of 5-formyltetrahydrofolate to 5,10-methenyltetrahydrofolate. Folate analogs that cannot undergo the rate-limiting step in catalysis were inhibitors of murine MTHFS. 5-Formyltetrahydrohomofolate was an effective inhibitor of murine MTHFS (K(i)=0.7 microM), whereas 5-formyl,10-methyltetrahydrofolate was a weak inhibitor (K(i)=10 microM). The former, but not the latter, was slowly phosphorylated by MTHFS. 5-Formyltetrahydrohomofolate was not a substrate for murine MTHFS, but was metabolized when the MTHFS active site Y151 was mutated to Ala. MTHFS active site residues do not directly facilitate N10 attack on the on the N5-iminium phosphate intermediate, but rather restrict N10 motion around N5. Inhibitors specifically designed to block N10 attack appear to be less effective than the natural 10-formyltetrahydrofolate polyglutamate inhibitors.

Airway hyperresponsiveness to histamine in mycoplasmal infection: role of histamine N-methyltransferase.

To elucidate the modulatory role of histamine-degrading enzymes in airway constrictor responses in mycoplasmal infection, we studied hamster tracheal segments under isometric conditions in vitro. Nasal inoculation with Mycoplasma pneumoniae potentiated the contractile responses to histamine but not to methacholine. Pretreatment of tissues with the histamine N-methyltransferase inhibitor SKF 91488 abolished the infection-induced potentiation, whereas, the diamine oxidase inhibitor aminoguanidine had no effect. The histamine N-methyltransferase but not diamine oxidase activity in tracheal tissues was decreased in infected animals. These results suggest that M. pneumoniae causes airway hyperresponsiveness to histamine probably through a reduction of endogenous histamine N-methyltransferase activity.

Adenosine deaminase activity in the aetiological diagnosis of community-acquired pneumonia.

A prospective study was undertaken to assess the usefulness of serum adenosine deaminase (ADA) activity in the aetiological diagnosis of 75 patients (mean age 58 years) with community-acquired pneumonia who required hospitalization. Measurements of ADA were also carried out in 35 healthy subjects (mean age 52 years). The serum ADA activity in patients with typical bacterial pneumonia (TBP) was 21 +/- 7 IU/l and in controls 22 +/- 9 IU/l. In 43 patients with atypical pneumonia (AP), ADA levels (43 +/- 23 IU/l) were significantly higher than in the previously related groups (p < 0.001). Analysis within the group of atypical pneumonia showed significant differences for infections caused by Coxiella burnetii (61 +/- 19 IU/l, p < 0.001), Mycoplasma pneumoniae (44 +/- 26 IU/l, p < 0.001) and Legionella pneumophila (39 +/- 15 IU/l, p < 0.05), as compared with patients with bacterial pneumonia and normal control subjects. We conclude that serum ADA in patients with community-acquired pneumonia requiring hospitalization may provide useful additional diagnostic information on the aetiology of pulmonary infection.

Expression of peroxidase activity in rat tracheal epithelial cells associated with Mycoplasma pulmonis.

Endogenous peroxidase activity has not been localized in the tracheal mucosal epithelial cells of specific pathogen-free (SPF) rats. After natural infection with Mycoplasma pulmonis in SPF rats, peroxidase activity became localized to the endoplasmic reticulum, nuclear envelope, and Golgi apparatus of tracheal ciliated or mucous secretory cells. Some secretory cells occasionally had peroxidase-positive secretory granules. At 1 wk M. pulmonis was found to attach to these epithelial cells, which then showed positive peroxidase activity at 2 wk. Serum antibody titers against M. pulmonis were positive at 5 wk. These results suggest that virulent mycoplasma infection and interaction with the tracheal epithelial cells trigger the de novo expression of peroxidase activity, which seems to play a role in mucosal anti-microbial defense mechanisms.

Adenosine deaminase activity and free IL-2 receptor levels in serum from patients with mycoplasma pneumonia.

Adenosine deaminase (ADA) activity and free interleukin (IL)-2 receptor levels were assayed in serum samples from patients with mycoplasma and bacterial pneumonia to evaluate the usefulness of these parameters in distinguishing between these diseases at an early stage. Serum ADA and free IL-2 receptor levels in patients with mycoplasma pneumonia (32.4 +/- 9.2 U/l, 960 +/- 204 U/ml) were significantly higher than those in patients with bacterial pneumonia (12.5 +/- 3.3 U/l, 425 +/- 86 U/ml) and in healthy controls (14.0 +/- 3.4 U/l, 286 +/- 49 U/ml) (p less than 0.001). Of the 20 mycoplasma pneumonia cases, 19 showed increased levels of ADA over 20.8 U/l; in 17 of the 19, the increase of ADA was seen before the elevation of the specific antibody to Mycoplasma pneumoniae. In contrast, serum ADA levels in all 20 cases of bacterial pneumonia were lower than 20.8 U/l. There results indicate that assays for serum ADA and free IL-2 receptor levels are useful in distinguishing between bacterial and mycoplasma pneumonia at an early stage.

Serum adenosine deaminase in viral and bacterial pneumonia.

We measured the activity of serum adenosine deaminase (ADA) in paired sera from 171 military conscripts with radiographically verified pneumonia. Patient serum samples were selected on the basis of serologic analyses identifying as single etiologic agents Streptococcus pneumoniae in 29 patients, Haemophilus influenzae in 7, Mycoplasma pneumoniae in 43, adenovirus in 24, influenza A or B in 12, and parainfluenza in 5 patients. In 14 patients Neisseria meningitidis and in 31 Chlamydia spp were considered the main etiologic agent. Compared with a control group of 45 healthy men, the ADA activity in patients with pneumonia was significantly higher (p less than 0.001) in all patient groups except those with meningococcal pneumonia. The highest ADA levels were seen in patients with pneumonia caused by M pneumoniae (27.4 +/- 9.7 U/L), Chlamydia spp (26.3 +/- 9.1 U/L), and adenovirus (28.5 +/- 10.9 U/L) compared with the controls (11.1 +/- 3.0 U/L). In patients with meningococcal pneumonia, the ADA activity was significantly decreased (p less than 0.001). Serum ADA activity probably reflects differences in cellular immune response to different infectious agents. The ADA determinations may give corroborative information on the etiologic agent of pneumonia.

Detection of elevated levels of 2-5A synthetase in serum from children with various infectious diseases.

By a sensitive radioimmunoassay method, (2'-5')oligoadenylate synthetase was detected in serum from patients with viral, bacterial, or mycoplasmal infections at elevated levels compared with enzyme levels in serum from healthy individuals and patients suffering from noninfectious diseases.