Roger Dumke

Occurrence of macrolide-resistant Mycoplasma pneumoniae strains in Germany

In a total of 167 respiratory tract specimens from adult outpatients with confirmed Mycoplasma pneumoniae pneumonia, sampled between 2003 and 2008, and a further 99 isolates obtained from patients between 1991 and 2009 in Germany, M. pneumoniae was tested for macrolide resistance. Using PCR, real-time PCR and sequencing of the 23S rRNA gene, 1.2% of M. pneumoniae in the respiratory tract samples and 3.0% of the isolates were found to be resistant. The results indicate a limited but not negligible importance of macrolide-resistant M. pneumoniae in the population investigated, which requires the monitoring of macrolide susceptibility of isolates or the testing of respiratory samples by molecular methods.

Culture-Independent Molecular Subtyping of Mycoplasma pneumoniae in Clinical Samples

ABSTRACT A new molecular subtyping approach was developed which is based on the amplification and sequencing of a repetitive region of the P1 gene of Mycoplasma pneumoniae. It allows the differentiation of all known subtypes and variants of M. pneumoniae as well as the identification of new subtypes directly in clinical samples to characterize endemic and epidemic M. pneumoniae infections.

Subtyping of Mycoplasma pneumoniae isolates based on extended genome sequencing and on expression profiles

Mycoplasma pneumoniae isolates from patients, collected over a period of 12 years in Germany, were characterized by various methods (parameters) including multilocus sequence typing, restriction fragment length polymorphisms, Western blotting with mono-specific antibodies directed against selected proteins or with polyspecific antibodies directed against the Triton X-114-soluble protein fraction, and two-dimensional gel electrophoresis. The results for 91 isolates from Germany, which were complemented with 14 isolates from the USA and 10 isolates from France, clearly showed that M. pneumoniae is a highly uniform species and that most of the isolates could be assigned to one of the two subtypes 1 and 2. The members of one subtype differ from the other with respect to the sequence of the P1 gene, the ORF6 gene, the P65 gene, and by a typical DNA restriction fragment pattern. We observed four isolates (variants), which seemed identical by the above mentioned criteria, but did not belong to either one of the two subtypes. They showed most of the subtype 2-specific features, but differed in the sequence of the P1 gene and showed a variation in the restriction fragment pattern. The appearance of subtype 1 or 2 over the last 12 years in Germany showed a dominance of subtype 1 between 1989 and 1996 and a dominance of subtype 2 between 1997 and 1998. The variant (neither subtype 1 nor subtype 2) was only detected in 1991 and 1995 but it had no epidemiological consequences.

Sensitive Detection of Mycoplasma pneumoniae in Human Respiratory Tract Samples by Optimized Real-Time PCR Approach

ABSTRACT To enhance the sensitivity of the available real-time PCR systems for the detection of Mycoplasma pneumoniae, we established a method to amplify copies of the repetitive element repMp1. In a study of respiratory tract samples, we found that, compared to the use of the conserved part of the P1 adhesin gene as a monocopy target, the use of the repMp1-PCR showed an increase in the detected genome equivalents by a factor of 22.

Identification of Risk Factors for Infection in an Outbreak of Mycoplasma pneumoniae Respiratory Tract Disease

BACKGROUND Mycoplasma pneumoniae is one of the most common pathogens that causes community-acquired respiratory tract infection. Outbreaks are well known, and all age groups are susceptible. An outbreak in an army training unit afforded an opportunity to identify possible risk factors for morbidity. METHODS An outbreak of respiratory illness that occurred in a unit comprising 91 trainees was investigated and analyzed as a cohort study. M. pneumoniae infection was suspected on clinical grounds and was confirmed by polymerase chain reaction, culture, and serologic testing. Data regarding medical history, symptoms, signs, and laboratory tests were collected. RESULTS During a period of 12 days, 41 soldiers (45.1%) had respiratory illnesses, of which 10 (11.0%) were pneumonia. Comparison of symptomatic and asymptomatic individuals revealed that smoking was associated with higher rates of disease (risk ratio, 2.1; 95% confidence interval [CI], 1.3-3.2; P<.005) and seroconversion (risk ratio, 2; 95% CI, 1.2-3.4; P=.03). In multivariate analysis, both lower acute immunoglobulin G values (adjusted odds ratio, 7.8; 95% CI, 1.4-42.5; P=.018) and smoking (adjusted odds ratio, 5.6; 95% CI, 1.5-20.4; P=.01) were associated with symptomatic infection; stratification according to smoking status revealed that immunoglobulin G levels among nonsmokers were protective. Patients who had pneumonia had lower lymphocyte counts (1400+/-258 vs. 2000+/-465 cells/microL; P=.001). CONCLUSIONS Smoking and lower preexisting immunoglobulin G levels were strongly associated with M. pneumoniae respiratory infection. These findings emphasize the importance of immunity and cessation of smoking for the prevention of disease. The high attack rate emphasizes the extent of infection transmission among healthy persons living in close contact.

Detection of phages carrying the Shiga toxin 1 and 2 genes in waste water and river water samples

Aims:  To evaluate the occurrence and abundance of phages that carry the stx1 and stx2 gene in water samples of different quality.

Mycoplasma pneumoniae and Chlamydia spp. Infection in Community-Acquired Pneumonia, Germany, 2011-2012

M. pneumoniae infections showed a strong epidemic peak, but Chlamydia spp. were consistently detected throughout the year.

Culture-independent multi-locus variable-number tandem-repeat analysis (MLVA) of Mycoplasma pneumoniae

A PCR approach for multi-locus variable-number tandem-repeat analysis (MLVA) of Mycoplasma pneumoniae directly from respiratory samples was developed and evaluated on 54 specimens from M. pneumoniae-positive pneumonia patients. The method resulted in a clearly identifiable MLVA type in all samples tested and can be used for MLVA without laborious isolation of the pathogen.

Characterization of pyruvate dehydrogenase subunit B and enolase as plasminogen-binding proteins in Mycoplasma pneumoniae.

The obligate pathogenic mycoplasma species Mycoplasma pneumoniae uses a limited but effective repertoire of virulence factors to infect and colonize the human respiratory tract. Besides the development of a unique adhesion complex and the expression of tissue-damaging factors, surface-located glycolytic enzymes and their capacity to bind to components of the human extracellular matrix (ECM) support pathogen-host interactions. Here, we demonstrated that the glycolytic enzymes enolase (Mpn606) and pyruvate dehydrogenase subunit B (Mpn392; PDHB) of M. pneumoniae show concentration-dependent binding to human plasminogen. Monospecific polyclonal antisera against both recombinant proteins reduced the binding to plasminogen significantly. The surface location of PDHB but not of enolase was demonstrated using Triton X fractionation of M. pneumoniae total protein content, membrane fractionation, colony blotting, mild proteolysis of mycoplasma cells, and immunofluorescence tests. To characterize the binding site of plasminogen in surface-displaced PDHB, the mycoplasmal protein was separated into four recombinant proteins followed by investigation of the binding behaviour of peptides that overlap the protein part interacting with plasminogen. Spot analysis resulted in a novel region of 12 amino acids (FPAMFQIFTHAA, position 91 to 102 of PDHB), which is responsible exclusively for binding of human plasminogen and also interacts in a dose-dependent manner with this host protein. The data indicate that the plasminogen-binding enzymes enolase and especially the surface-associated PDHB may contribute to the pathogenesis of M. pneumoniae infections.

Subtypes and variants of Mycoplasma pneumoniae : local and temporal changes in Germany 2003–2006 and absence of a correlation between the genotype in the respiratory tract and the occurrence of genotype-specific antibodies in the sera of infected patients

Mycoplasma pneumoniae is a frequent cause of community-acquired pneumonia. Three subtypes and three variants of M. pneumoniae have been described showing sequence differences in the main P1 adhesin. Between 2003 and 2006 we collected respiratory tract samples of adult outpatients with symptoms of pneumonia in a German nationwide network and detected M. pneumoniae by real-time PCR in 140 specimens. The strains were typed by sequencing and demonstrated the circulation of subtypes 1 and 2 and variants 2a and 2b. The overall number of isolates belonging to the two variant genotypes increased during the investigation period but the relationship of subtypes and variants within the participating local centres varied strongly. ELISA experiments using sera of acute-phase patients with a known M. pneumoniae type in the respiratory tract resulted in no correlation of IgA and IgG antibodies to subtype- and variant-specific regions of the P1 gene with the genotype of the M. pneumoniae strain causing the actual infection.