Regina Konrad

Outbreaks of Pneumocystis Pneumonia in 2 Renal Transplant Centers Linked to a Single Strain of Pneumocystis: Implications for Transmission and Virulence

BACKGROUND There have been numerous reports of clustered outbreaks of Pneumocystis pneumonia (PCP) at renal transplant centers over the past 2 decades. It has been unclear whether these outbreaks were linked epidemiologically to 1 or several unique strains, which could have implications for transmission patterns or strain virulence. METHODS Restriction fragment length polymorphism (RFLP) analysis was used to compare Pneumocystis isolates from 3 outbreaks of PCP in renal transplant patients in Germany, Switzerland, and Japan, as well as nontransplant isolates from both human immunodeficiency virus (HIV)-infected and uninfected patients. RESULTS Based on RFLP analysis, a single Pneumocystis strain caused pneumonia in transplant patients in Switzerland (7 patients) and Germany (14 patients). This strain was different from the strain that caused an outbreak in transplant patients in Japan, as well as strains causing sporadic cases of PCP in nontransplant patients with or without HIV infection. CONCLUSIONS Two geographically distinct clusters of PCP in Europe were due to a single strain of Pneumocystis. This suggests either enhanced virulence of this strain in transplant patients or a common, but unidentified, source of transmission. Outbreaks of PCP can be better understood by enhanced knowledge of transmission patterns and strain variation.

Application of MALDI-TOF MS for the Identification of Food Borne Bacteria

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has recently emerged as a powerful tool for the routine identification of clinical isolates. MALDI-TOF MS based identification of bacteria has been shown to be more rapid, accurate and cost-efficient than conventional phenotypic techniques or molecular methods. Rapid and reliable identification of food-associated bacteria is also of crucial importance for food processing and product quality. This review is concerned with the applicability of MALDI-TOF MS for routine identification of foodborne bacteria taking the specific requirements of food microbiological laboratories and the food industry into account. The current state of knowledge including recent findings and new approaches are discussed.

Leptospira spp. strain identification by MALDI TOF MS is an equivalent tool to 16S rRNA gene sequencing and multi locus sequence typing (MLST)

BackgroundIn this study mass spectrometry was used for evaluating extracted leptospiral protein samples and results were compared with molecular typing methods. For this, an extraction protocol for Leptospira spp. was independently established in two separate laboratories. Reference spectra were created with 28 leptospiral strains, including pathogenic, non-pathogenic and intermediate strains. This set of spectra was then evaluated on the basis of measurements with well-defined, cultured leptospiral strains and with 16 field isolates of veterinary or human origin. To verify discriminating peaks for the applied pathogenic strains, statistical analysis of the protein spectra was performed using the software tool ClinProTools. In addition, a dendrogram of the reference spectra was compared with phylogenetic trees of the 16S rRNA gene sequences and multi locus sequence typing (MLST) analysis.ResultsDefined and reproducible protein spectra using MALDI-TOF MS were obtained for all leptospiral strains. Evaluation of the newly-built reference spectra database allowed reproducible identification at the species level for the defined leptospiral strains and the field isolates. Statistical analysis of three pathogenic genomospecies revealed peak differences at the species level and for certain serovars analyzed in this study. Specific peak patterns were reproducibly detected for the serovars Tarassovi, Saxkoebing, Pomona, Copenhageni, Australis, Icterohaemorrhagiae and Grippotyphosa. Analysis of the dendrograms of the MLST data, the 16S rRNA sequencing, and the MALDI-TOF MS reference spectra showed comparable clustering.ConclusionsMALDI-TOF MS analysis is a fast and reliable method for species identification, although Leptospira organisms need to be produced in a time-consuming culture process. All leptospiral strains were identified, at least at the species level, using our described extraction protocol. Statistical analysis of the three genomospecies L. borgpetersenii, L. interrogans and L. kirschneri revealed distinctive, reproducible differentiating peaks for seven leptospiral strains which represent seven serovars. Results obtained by MALDI-TOF MS were confirmed by MLST and 16S rRNA gene sequencing.

German Francisella tularensis isolates from European brown hares (Lepus europaeus)reveal genetic and phenotypic diversity

BackgroundTularemia is a zoonotic disease caused by Francisella tularensis that has been found in many different vertebrates. In Germany most human infections are caused by contact with infected European brown hares (Lepus europaeus). The aim of this study was to elucidate the epidemiology of tularemia in hares using phenotypic and genotypic characteristics of F. tularensis.ResultsCultivation of F. tularensis subsp. holarctica bacteria from organ material was successful in 31 of 52 hares that had a positive PCR result targeting the Ft-M19 locus. 17 isolates were sensitive to erythromycin and 14 were resistant. Analysis of VNTR loci (Ft-M3, Ft-M6 and Ft-M24), INDELs (Ftind33, Ftind38, Ftind49, RD23) and SNPs (B.17, B.18, B.19, and B.20) was shown to be useful to investigate the genetic relatedness of Francisella strains in this set of strains. The 14 erythromycin resistant isolates were assigned to clade B.I, and 16 erythromycin sensitive isolates to clade B.IV and one isolate was found to belong to clade B.II. MALDI-TOF mass spectrometry (MS) was useful to discriminate strains to the subspecies level.ConclusionsF. tularensis seems to be a re-emerging pathogen in Germany. The pathogen can easily be identified using PCR assays. Isolates can also be identified within one hour using MALDI-TOF MS in laboratories where specific PCR assays are not established. Further analysis of strains requires genotyping tools. The results from this study indicate a geographical segregation of the phylogenetic clade B.I and B.IV, where B.I strains localize primarily within eastern Germany and B.IV strains within western Germany. This phylogeographical pattern coincides with the distribution of biovar I (erythromycin sensitive) and biovar II (erythromycin resistance) strains. When time and costs are limiting parameters small numbers of isolates can be analysed using PCR assays combined with DNA sequencing with a focus on genetic loci that are most likely discriminatory among strains found in a specific area. In perspective, whole genome data will have to be investigated especially when terrorist attack strains need to be tracked to their genetic and geographical sources.

Identification and differentiation of food-related bacteria: A comparison of FTIR spectroscopy and MALDI-TOF mass spectrometry.

The food industry requires easy, accurate, and cost-effective techniques for microbial identification to ensure safe products and identify microbial contaminations. In this work, FTIR spectroscopy and MALDI-TOF mass spectrometry were assessed for their suitability and applicability for routine microbial diagnostics of food-related microorganisms by analyzing their robustness according to changes in incubation time and medium, identification accuracy and their ability to differentiate isolates down to the strain level. Changes in the protocol lead to a significantly impaired performance of FTIR spectroscopy, whereas they had only little effects on MALDI-TOF MS. Identification accuracy was tested using 174 food-related bacteria (93 species) from an in-house strain collection and 40 fresh isolates from routine food analyses. For MALDI-TOF MS, weaknesses in the identification of bacilli and pseudomonads were observed; FTIR spectroscopy had most difficulties in identifying pseudomonads and enterobacteria. In general, MALDI-TOF MS obtained better results (52-85% correct at species level), since the analysis of mainly ribosomal proteins is more robust and seems to be more reliable. FTIR spectroscopy suffers from the fact that it generates a whole-cell fingerprint and intraspecies diversity may lead to overlapping species borders which complicates identification. In the present study values between 56% and 67% correct species identification were obtained. On the opposite, this high sensitivity offers the opportunity of typing below the species level which was not possible using MALDI-TOF MS. Using fresh isolates from routine diagnostics, both techniques performed well with 88% (MALDI-TOF) and 75% (FTIR) correct identifications at species level, respectively.

Next generation sequencing analysis of nine Corynebacterium ulcerans isolates reveals zoonotic transmission and a novel putative diphtheria toxin-encoding pathogenicity island.

BackgroundToxigenic Corynebacterium ulcerans can cause a diphtheria-like illness in humans and have been found in domestic animals, which were suspected to serve as reservoirs for a zoonotic transmission. Additionally, toxigenic C. ulcerans were reported to take over the leading role in causing diphtheria in the last years in many industrialized countries.MethodsTo gain deeper insights into the tox gene locus and to understand the transmission pathway in detail, we analyzed nine isolates derived from human patients and their domestic animals applying next generation sequencing and comparative genomics.ResultsWe provide molecular evidence for zoonotic transmission of C. ulcerans in four cases and demonstrate the superior resolution of next generation sequencing compared to multi-locus sequence typing for epidemiologic research. Additionally, we provide evidence that the virulence of C. ulcerans can change rapidly by acquisition of novel virulence genes. This mechanism is exemplified by an isolate which acquired a prophage not present in the corresponding isolate from the domestic animal. This prophage contains a putative novel virulence factor, which shares high identity with the RhuM virulence factor from Salmonella enterica but which is unknown in Corynebacteria so far. Furthermore, we identified a putative pathogenicity island for C. ulcerans bearing a diphtheria toxin gene.ConclusionThe novel putative diphtheria toxin pathogenicity island could provide a new and alternative pathway for Corynebacteria to acquire a functional diphtheria toxin-encoding gene by horizontal gene transfer, distinct from the previously well characterized phage infection model. The novel transmission pathway might explain the unexpectedly high number of toxigenic C. ulcerans.

Development of a Multiplex Real-Time Polymerase Chain Reaction for Simultaneous Detection of Enterohemorrhagic Escherichia coli and Enteropathogenic Escherichia coli Strains

A multiplex real-time polymerase chain reaction (PCR) was developed for the simultaneous detection of genes encoding intimin (eae) and all variants of Shiga toxins 1 and 2 (stx1 and stx2) in diagnostic samples. The uidA gene encoding a beta-glucuronidase specific for Escherichia coli and Shigella spp. was included in the multiplex PCR assay as an internal amplification control. The multiplex PCR was tested on 30 E. coli reference strains and 174 diagnostic samples already characterized as harboring stx1, stx2, and eae genes. The multiplex PCR correctly detected the genes in all strains examined. No cross reaction was observed with 68 strains representing other gastrointestinal pathogens, normal gastrointestinal flora, or closely related bacteria, reflecting 100% specificity of the assay. The detection limits of the multiplex PCR were 5 genome equivalents for stx2 and 50 genome equivalents for eae and stx1.

Multilocus Sequence Typing of Corynebacterium ulcerans Provides Evidence for Zoonotic Transmission and for Increased Prevalence of Certain Sequence Types among Toxigenic Strains

ABSTRACT Human-to-human-transmitted Corynebacterium diphtheriae was historically the main pathogen causing diphtheria and has therefore been studied extensively in the past. More recently, diphtheria caused by toxigenic Corynebacterium ulcerans is an emerging disease in several industrial countries, including the United Kingdom, the United States, France, and Germany. However, toxigenic C. ulcerans has so far been almost neglected in the development of epidemiologic tools. One of the most important tools in modern epidemiology to understand transmission pathways is sequence typing of pathogens. Here, we provide a protocol for multilocus sequence typing (MLST) to type C. ulcerans strains rapidly and relatively cost-effectively. Applying MLST to C. ulcerans for the first time, we show that related sequence types (STs) might be associated with the presence of the diphtheria toxin gene, which encodes diphtheria toxin (DT), the most important diphtheria-causing virulence factor. Interestingly, we found only two very closely related STs in the isolates derived from six dogs. Additionally, our data show that all STs derived from animals which were at least twice present in our analysis were found in humans as well. This finding is congruent with zoonotic transmission of C. ulcerans.

Zoonotic Transmission of Toxigenic Corynebacterium ulcerans Strain, Germany, 2012

Severe necrotizing fasciitis was diagnosed in a 53-year-old man in Germany in 2012. Toxigenic Corynebacterium ulcerans was grown from a wound swab sample. One of the patient´s 2 dogs was found to harbor a toxigenic C. ulcerans strain. Results of next generation sequencing of both isolates supported recent zoonotic transmission of this bacterial pathogen.

Detection Methods for Laboratory Diagnosis of Diphtheria

Although diphtheria has to be diagnosed primarily on clinical symptoms, the rapid and reliable detection and identification of the potentially toxigenic Corynebacterium species, C. diphtheriae, C. ulcerans and C. pseudotuberculosis, is essential for the definite diagnosis and management of diphtheria with respect to both the individual patient and the public health measures to be undertaken. Laboratory confirmation of suspected diphtheria has to aim for the isolation of the etiologic pathogen (including species identification and antibiotic susceptibility testing) as well as for differentiation of toxigenic from non-toxigenic strains (by using tox gene detection and toxigenicity testing). The recent introduction of the Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) into the microbiological algorithm of laboratory diagnosis of diphtheria allows the specific and escalating identification of the three potentially toxigenic Corynebacterium species from growing colonies. Finally, molecular typing techniques may be applied to explore the clonal relatedness of clinical isolates and their potential routes of transmission. The most relevant laboratory procedures fulfilling these requirements (microbiological culture, conventional biochemical tests, molecular methods for species identification and toxigenicity testing) will be presented here.