Paul Christian Lück

Addition of neuA, the Gene Encoding N-Acylneuraminate Cytidylyl Transferase, Increases the Discriminatory Ability of the Consensus Sequence-Based Scheme for Typing Legionella pneumophila Serogroup 1 Strains

ABSTRACT The standard sequence-based method for the typing of Legionella pneumophila serogroup 1 strains was extended by using the gspA and neuA alleles. The use of neuA as a seventh allele for typing significantly increased the index of discrimination calculated for a panel of unrelated strains (from 0.932 to 0.963) and subdivided some known large common complexes (e.g., 1,4,3,1,1,1). This modification to the standard method is proposed as the method of choice in the epidemiological investigation of L. pneumophila infections.

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.

Molecular characterization of a virulence-associated epitope on the lipopolysaccharide of Legionella pneumophila serogroup 1.

For identification of lipopolysaccharide (LPS)-associated epitopes of Legionella pneumophila serogroup 1, LPS of strain Philadelphia 1 was investigated using monoclonal antibodies (MAbs). The O-specific chain of LPS is a homopolymer of 5-acetamidino-7-acetamido-8-O-acetyl-3,5,7,9-tetradeoxy-D-glycero- L-galacto- nonulosonic acid. At least four immunoaccessible epitopes were recognized by different MAbs on the intact LPS. After O-deacetylation of LPS, the reactivity of one of the MAbs (MAb 3/1) was lost, indicating thus that the corresponding epitope is associated with the 8-O-acetyl group. Since the reactivity pattern of the MAb 3/1 is identical with those of the MAb 2 which was considered as a virulence marker for serogroup 1, this epitope may be involved in mediating virulence in L. pneumophila. Four MAbs specific to strains of serogroup 1 other than the monoclonal subtype Philadelphia recognized epitopes on the O-deacetylated LPS of strain Philadelphia 1 and, therefore, the virulence-associated epitope blocks recognition of the immunodeterminants that are accessible on the intact LPS of the strains lacking this epitope.

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.

Occurrence and distribution of sequence types among Legionella pneumophila strains isolated from patients in Germany: common features and differences to other regions of the world

A total of 105 unrelated clinical isolates of Legionella pneumophila were randomly selected from the German National Legionella strain collection and typed by monoclonal antibody (MAb) subgrouping and a seven-gene locus sequence-based typing (SBT) scheme. According to the case definitions of the European Working Group for Legionella Infections, 19 of the isolates tested were travel-associated, 38 were community-acquired and 48 were of nosocomial origin. Eighty-four of these strains belonged to serogroup 1, 20 belonged to other serogroups, and one isolate could not be serogrouped. The majority of strains among the travel-associated and community-acquired cases were MAb3-1-positive. The most common sequence type (1, 4, 3, 1, 1, 1, 1) was found in 20 isolates in 11 cities; other allelic profiles also found in Europe (2, 3, 9, 10, 2, 1, 6), (1, 3, 9, 10, 2, 1, 6), (2, 6, 17, 14, 13, 11, 11) and (3, 4, 1, 1, 1, 9, 1) were detected among the German isolates but at a low frequency. In contrast, some SBT are unique to Germany, including (3, 4, 1, 3, 35, 9, 11), which was found among five isolates from patients in Berlin. In concordance with European data, a significant portion of the L. pneumophila strains isolated from patients in Germany belong to clones that occur throughout the world and which are responsible for the majority of clinical cases.

Diagnostic Relevance of the Detection of Legionella DNA in Urine Samples by the Polymerase Chain Reaction

Abstract Urine samples from 317 patients with pneumonia and from 242 patients without pneumonia were tested using a polymerase chain reaction (PCR) system for detection of the Legionella 5S rRNA gene. The results were compared with findings obtained using the established methods for diagnosis of legionellosis. Of the 317 patients with pneumonia, 58 had confirmed legionellosis, 35 had a presumptive Legionella infection, and 224 had no evidence of Legionella infection as determined by conventional methods using published criteria. The PCR was positive for 42 patients with confirmed infections, yielding a sensitivity of 72.4%. Furthermore, 16 (47%) patients with presumptive legionellosis and five (2.2%) patients without other evidence of Legionella infection had positive results. All samples from 242 patients without pneumonia were PCR-negative. When the results for all patients were considered, the specificity of the assay was ≥98.9%. The results demonstrate that the sensitivity and specificity values of urinary PCR are in the same range as those of established methods. The use of PCR in urine complements the repertoire of rapid diagnostic methods, especially for infections caused by legionellae not belonging to Legionella pneumophila serogroup 1, in which tests for detection of urinary antigen often fail.

Immunolocalization of the Mip protein of intracellularly and extracellularly grown Legionella pneumophila.

J.H. HELBIG, P.C. LÜCK, M. STEINERT, E. JACOBS AND M. WITT. 2001. The macrophage infectivity potentiator (Mip) protein is an important factor in the optimal intracellular survival of Legionella pneumophila in protozoa and human cell lines. In this study we have localized the Mip protein in Legionella grown on buffered charcoal yeast extract (BCYE) agar as well as in Legionella which were ingested by Acanthamoeba castellanii. Immunogold techniques have shown that Mip is exposed on the cell surface of extracellularly grown bacteria. In A. castellanii infected with Legionella the Mip protein was also detected on host membranes which exhibited a multilamellar structure. The morphology of these structures is similar to that of respirable vesicles of amoebas by which live legionellas may be transmitted to humans. It can be assumed that the accumulation of Mip protein in the multilamellar host membranes increases the infection potential.

A Hospital-Associated Outbreak of Legionnaires' Disease Caused by Legionella pneumophila Serogroup 1 Is Characterized by Stable Genetic Fingerprinting but Variable Monoclonal Antibody Patterns

ABSTRACT An outbreak of 18 pneumonia cases caused by Legionella pneumophila serogroup 1 occurred at a Swedish university hospital 1996 to 1999. Eight clinical isolates obtained by culture from the respiratory tract were compared to 20 environmental isolates from the hospital and to 21 epidemiologically unrelated isolates in Sweden, mostly from patients, by using pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphism analysis (AFLP), and monoclonal antibody (MAb) typing. All patients and most environmental isolates from the outbreak hospital belonged to the same genotypic cluster in both PFGE and AFLP. This genotype was distinctly different from other strains, including a cluster from a second hospital in a different part of the country. The MAb subtype of the outbreak clone was Knoxville except for three isolates that were Oxford. A variation in the MAb reactivity pattern was also found in a second genotypic cluster. These changes in the MAb reactivity pattern were due to the absence or presence of the lag-1 gene coding for an O-acetyltransferase that is responsible for expression of the lipopolysaccharide epitope recognized by MAb 3/1 of the Dresden Panel. In all MAb 3/1-positive strains, the lag-1 gene was present on a genetic element that was bordered by a direct repeat that showed a high degree of sequence homology. Due to this homology, the lag-1 gene region seemed to be an unstable element in the chromosome. MAb patterns are thus a valuable adjunct to genotyping methods in defining subgroups inside a genotypic cluster of L. pneumophila sg 1.

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.

Identification and serotyping of atypical Legionella pneumophila strains isolated from human and environmental sources.

Aims:  To validate identification methods for Legionella pneumophila strains that cannot be serotyped into the known serogroups and to characterize their antigenic diversity.