Proteotyping as alternate typing method to differentiate Campylobacter coli clades.

Besides Campylobacter jejuni, Campylobacter coli is the most common bacterial cause of gastroenteritis worldwide. C. coli is subdivided into three clades, which are associated with sample source. Clade 1 isolates are associated with acute diarrhea in humans whereas clade 2 and 3 isolates are more commonly obtained from environmental waters. The phylogenetic classification of an isolate is commonly done using laborious multilocus sequence typing (MLST). The aim of this study was to establish a proteotyping scheme using MALDI-TOF MS to offer an alternative to sequence-based methods. A total of 97 clade-representative C. coli isolates were analyzed by MALDI-TOF-based intact cell mass spectrometry (ICMS) and evaluated to establish a C. coli proteotyping scheme. MLST was used as reference method. Different isoforms of the detectable biomarkers, resulting in biomarker mass shifts, were associated with their amino acid sequences and included into the C. coli proteotyping scheme. In total, we identified 16 biomarkers to differentiate C. coli into the three clades and three additional sub-clades of clade 1. In this study, proteotyping has been successfully adapted to C. coli. The established C. coli clades and sub-clades can be discriminated using this method. Especially the clinically relevant clade 1 isolates can be differentiated clearly.

Association of Campylobacter Jejuni ssp. Jejuni Chemotaxis Receptor Genes with Multilocus Sequence Types and Source of Isolation.

Campylobacter jejuni's flagellar locomotion is controlled by eleven chemoreceptors. Assessment of the distribution of the relevant chemoreceptor genes in the C. jejuni genomes deposited in the National Center for Biotechnology Information (NCBI) database led to the identification of two previously unknown tlp genes and a tlp5 pseudogene. These two chemoreceptor genes share the same locus in the C. jejuni genome with tlp4 and tlp11, but the gene region encoding the periplasmic ligand binding domain differs significantly from other chemoreceptor genes. Hence, they were named tlp12 and tlp13. Consequently, it was of interest to study their distribution in C. jejuni subpopulations of different clonality, and their cooccurrence with the eleven previously reported chemoreceptor genes. Therefore, the presence of all tlp genes was detected by polymerase chain reaction (PCR) in 292 multilocus sequence typing (MLST)-typed C. jejuni isolates from different hosts. The findings show interesting trends: Tlp4, tlp11, tlp12, and tlp13 appeared to be mutually exclusive and cooccur in a minor subset of isolates. Tlp4 was found to be present in only 33.56% of all tested isolates and was significantly less often detected in turkey isolates. Tlp11 was tested positive in only 17.8% of the isolates, while tlp12 was detected in 29.5% of all isolates, and tlp13 was found to be present in 38.7%.

Seroprevalence of Campylobacter-Specific Antibodies in two German Duck Farms - A Prospective Follow-Up Study.

Several studies have shown that about 60-100% of farmed ducks are colonized by Campylobacter species. Because of this, a higher risk of campylobacteriosis among duck farm workers can be assumed. To estimate the risk of Campylobacter infections in duck farm workers, we investigated the prevalence of Campylobacter spp. in ducks of two duck farms and the seroprevalence of anti-Campylobacter antibodies (IgA and IgG) in two cohorts of workers. The first cohort consisted of high-exposed stable workers and slaughterers, which was compared to a second cohort of non-/low-exposed persons. Duck caecal swabs and serum samples were collected in 2004, 2007, and 2010. The colonization rate in the examined ducks was found to be 80-90%. The seroprevalence of anti-Campylobacter IgA and IgG antibodies among the non-exposed cohort was found to be 0.00% in all 3 years. In contrast, the exposed cohort demonstrated an IgA seroprevalence of 4.17% in 2004, 5.71% in 2007, and 0.00% in 2010 and an IgG seroprevalence of 8.33% in 2004, 0.00% in 2007, and 4.29% in 2010. In conclusion, in 2004, we observed a significantly higher anti-Campylobacter antibody seroprevalence in the exposed cohort followed by a steady reduction in 2007 and 2010 under occupational health and safety measures.

Mass Spectrometry-based PhyloProteomics (MSPP): A novel microbial typing Method.

MALDI-TOF-MS of microorganisms, which identifies microbes based on masses of high abundant low molecular weight proteins, is rapidly advancing to become another standard method in clinical routine laboratory diagnostics. Allelic isoforms of these proteins result in varying masses of detectable biomarker ions. These variations give rise to a novel typing method for microorganisms named mass spectrometry-based phyloproteomics (MSPP). The base of MSPP is an amino acid sequence list of allelic isoforms caused by non-synonymous mutations in biomarker genes, which were detectable as mass shifts in an overlay of calibrated MALDI-TOF spectra. Thus, for each isolate a combination of amino acid sequences can be deduced from the scheme of recordable biomarker masses. Performing comparably to laborious multilocus and whole genome sequence typing (wgMLST)-approaches it is feasible to build phyloproteomic dendrograms using hierarchical cluster analysis. MSPP bears a high potential especially for identification of chromosomal localised virulence or antimicrobial resistance factors associated with evolutionary relatedness. In this study the principle of MSPP-typing was demonstrated on a Campylobacter jejuni ssp. jejuni isolate collection and MSPP was compared to MLST.

Infectious causes of cholesteatoma and treatment of infected ossicles prior to reimplantation by hydrostatic high-pressure inactivation.

Chronic inflammation, which is caused by recurrent infections, is one of the factors contributing to the pathogenesis of cholesteatoma. If reimplantation of autologous ossicles after a surgical intervention is intended, inactivation of planktonic bacteria and biofilms is desirable. High hydrostatic pressure treatment is a procedure, which has been used to inactivate cholesteatoma cells on ossicles. Here we discuss the potential inactivating effect of high hydrostatic pressure on microbial pathogens including biofilms. Recent experimental data suggest an incomplete inactivation at a pressure level, which is tolerable for the bone substance of ossicles and results at least in a considerable reduction of pathogen load. Further studies are necessary to access how far this quantitative reduction of pathogens is sufficient to prevent ongoing chronic infections, for example, due to forming of biofilms.

Emerging rapid resistance testing methods for clinical microbiology laboratories and their potential impact on patient management.

Atypical and multidrug resistance, especially ESBL and carbapenemase expressing Enterobacteriaceae, is globally spreading. Therefore, it becomes increasingly difficult to achieve therapeutic success by calculated antibiotic therapy. Consequently, rapid antibiotic resistance testing is essential. Various molecular and mass spectrometry-based approaches have been introduced in diagnostic microbiology to speed up the providing of reliable resistance data. PCR- and sequencing-based approaches are the most expensive but the most frequently applied modes of testing, suitable for the detection of resistance genes even from primary material. Next generation sequencing, based either on assessment of allelic single nucleotide polymorphisms or on the detection of nonubiquitous resistance mechanisms might allow for sequence-based bacterial resistance testing comparable to viral resistance testing on the long term. Fluorescence in situ hybridization (FISH), based on specific binding of fluorescence-labeled oligonucleotide probes, provides a less expensive molecular bridging technique. It is particularly useful for detection of resistance mechanisms based on mutations in ribosomal RNA. Approaches based on MALDI-TOF-MS, alone or in combination with molecular techniques, like PCR/electrospray ionization MS or minisequencing provide the fastest resistance results from pure colonies or even primary samples with a growing number of protocols. This review details the various approaches of rapid resistance testing, their pros and cons, and their potential use for the diagnostic laboratory.

Modification of intestinal microbiota and its consequences for innate immune response in the pathogenesis of campylobacteriosis.

Campylobacter jejuni is the leading cause of bacterial food-borne gastroenteritis in the world, and thus one of the most important public health concerns. The initial stage in its pathogenesis after ingestion is to overcome colonization resistance that is maintained by the human intestinal microbiota. But how it overcomes colonization resistance is unknown. Recently developed humanized gnotobiotic mouse models have provided deeper insights into this initial stage and host's immune response. These studies have found that a fat-rich diet modifies the composition of the conventional intestinal microbiota by increasing the Firmicutes and Proteobacteria loads while reducing the Actinobacteria and Bacteroidetes loads creating an imbalance that exposes the intestinal epithelial cells to adherence. Upon adherence, deoxycholic acid stimulates C. jejuni to synthesize Campylobacter invasion antigens, which invade the epithelial cells. In response, NF- κ B triggers the maturation of dendritic cells. Chemokines produced by the activated dendritic cells initiate the clearance of C. jejuni cells by inducing the actions of neutrophils, B-lymphocytes, and various subsets of T-cells. This immune response causes inflammation. This review focuses on the progress that has been made on understanding the relationship between intestinal microbiota shift, establishment of C. jejuni infection, and consequent immune response.

Discrimination of multilocus sequence typing-based Campylobacter jejuni subgroups by MALDI-TOF mass spectrometry.

BACKGROUND: Campylobacter jejuni, the most common bacterial pathogen causing gastroenteritis, shows a wide genetic diversity. Previously, we demonstrated by the combination of multi locus sequence typing (MLST)-based UPGMA-clustering and analysis of 16 genetic markers that twelve different C. jejuni subgroups can be distinguished. Among these are two prominent subgroups. The first subgroup contains the majority of hyperinvasive strains and is characterized by a dimeric form of the chemotaxis-receptor Tlp7(m+c). The second has an extended amino acid metabolism and is characterized by the presence of a periplasmic asparaginase (ansB) and gamma-glutamyl-transpeptidase (ggt). RESULTS: Phyloproteomic principal component analysis (PCA) hierarchical clustering of MALDI-TOF based intact cell mass spectrometry (ICMS) spectra was able to group particular C. jejuni subgroups of phylogenetic related isolates in distinct clusters. Especially the aforementioned Tlp7(m+c)(+) and ansB+/ ggt+ subgroups could be discriminated by PCA. Overlay of ICMS spectra of all isolates led to the identification of characteristic biomarker ions for these specific C. jejuni subgroups. Thus, mass peak shifts can be used to identify the C. jejuni subgroup with an extended amino acid metabolism. CONCLUSIONS: Although the PCA hierarchical clustering of ICMS-spectra groups the tested isolates into a different order as compared to MLST-based UPGMA-clustering, the isolates of the indicator-groups form predominantly coherent clusters. These clusters reflect phenotypic aspects better than phylogenetic clustering, indicating that the genes corresponding to the biomarker ions are phylogenetically coupled to the tested marker genes. Thus, PCA clustering could be an additional tool for analyzing the relatedness of bacterial isolates.