Comparative analysis of proteomic adaptations in Enterococcus faecalis and Enterococcus faecium after long term bile acid exposure.

BACKGROUND: All gastrointestinal pathogens, including Enterococcus faecalis and Enterococcus faecium, undergo adaptation processes during colonization and infection. In this study, we investigated by data-independent acquisition mass spectrometry (DIA-MS) two crucial adaptations of these two Enterococcus species at the proteome level. Firstly, we examined the adjustments to cope with bile acid concentrations at 0.05% that the pathogens encounter during a potential gallbladder infection. Therefore, we chose the primary bile acids cholic acid (CA) and chenodeoxycholic acid (CDCA) as well as the secondary bile acid deoxycholic acid (DCA), as these are the most prominent bile acids. Secondly, we investigated the adaptations from an aerobic to a microaerophilic environment, as encountered after oral-fecal infection, in the absence and presence of deoxycholic acid (DCA). RESULTS: Our findings showed similarities, but also species-specific variations in the response to the different bile acids. Both Enterococcus species showed an IC50 in the range of 0.01- 0.023% for DCA and CDCA in growth experiments and both species were resistant towards 0.05% CA. DCA and CDCA had a strong effect on down-expression of proteins involved in translation, transcription and replication in E. faecalis (424 down-expressed proteins with DCA, 376 down-expressed proteins with CDCA) and in E. faecium (362 down-expressed proteins with DCA, 391 down-expressed proteins with CDCA). Proteins commonly significantly altered in their expression in all bile acid treated samples were identified for both species and represent a "general bile acid response". Among these, various subunits of a V-type ATPase, different ABC-transporters, multi-drug transporters and proteins related to cell wall biogenesis were up-expressed in both species and thus seem to play an essential role in bile acid resistance. Most of the differentially expressed proteins were also identified when E. faecalis was incubated with low levels of DCA at microaerophilic conditions instead of aerobic conditions, indicating that adaptations to bile acids and to a microaerophilic atmosphere can occur simultaneously. CONCLUSIONS: Overall, these findings provide a detailed insight into the proteomic stress response of two Enterococcus species and help to understand the resistance potential and the stress-coping mechanisms of these important gastrointestinal bacteria.

Characterization of Campylobacter jejuni proteome profiles in co-incubation scenarios.

In dynamic microbial ecosystems, bacterial communication is a relevant mechanism for interactions between different microbial species. When C. jejuni resides in the intestine of either avian or human hosts, it is exposed to diverse bacteria from the microbiome. This study aimed to reveal the influence of co-incubation with Enterococcus faecalis, Enterococcus faecium, or Staphylococcus aureus on the proteome of C. jejuni 81-176 using data-independent-acquisition mass spectrometry (DIA-MS). We compared the proteome profiles during co-incubation with the proteome profile in response to the bile acid deoxycholate (DCA) and investigated the impact of DCA on proteomic changes during co-incubation, as C. jejuni is exposed to both factors during colonization. We identified 1,375 proteins by DIA-MS, which is notably high, approaching the theoretical maximum of 1,645 proteins. S. aureus had the highest impact on the proteome of C. jejuni with 215 up-regulated and 230 down-regulated proteins. However, these numbers are still markedly lower than the 526 up-regulated and 516 down-regulated proteins during DCA exposure. We identified a subset of 54 significantly differentially expressed proteins that are shared after co-incubation with all three microbial species. These proteins were indicative of a common co-incubation response of C. jejuni. This common proteomic response partly overlapped with the DCA response; however, several proteins were specific to the co-incubation response. In the co-incubation experiment, we identified three membrane-interactive proteins among the top 20 up-regulated proteins. This finding suggests that the presence of other bacteria may contribute to increased adherence, e.g., to other bacteria but eventually also epithelial cells or abiotic surfaces. Furthermore, a conjugative transfer regulon protein was typically up-expressed during co-incubation. Exposure to both, co-incubation and DCA, demonstrated that the two stressors influenced each other, resulting in a unique synergistic proteomic response that differed from the response to each stimulus alone. Data are available via ProteomeXchange with identifier PXD046477.

Comparison of the Diagnostic Accuracy of Three Real-Time PCR Assays for the Detection of Arcobacter butzleri in Human Stool Samples Targeting Different Genes in a Test Comparison without a Reference Standard.

Potential etiological relevance for gastroenteric disorders including diarrhea has been assigned to Arcobacter butzleri. However, standard routine diagnostic algorithms for stool samples of patients with diarrhea are rarely adapted to the detection of this pathogen and so, A. butzleri is likely to go undetected unless it is specifically addressed, e.g., by applying pathogen-specific molecular diagnostic approaches. In the study presented here, we compared three real-time PCR assays targeting the genes hsp60, rpoB/C (both hybridization probe assays) and gyrA (fluorescence resonance energy transfer assay) of A. butzleri in a test comparison without a reference standard using a stool sample collection with a high pretest probability from the Ghanaian endemicity setting. Latent class analysis was applied with the PCR results obtained with a collection of 1495 stool samples showing no signs of PCR inhibition to assess the real-time PCR assays' diagnostic accuracy. Calculated sensitivity and specificity were 93.0% and 96.9% for the hsp60-PCR, 100% and 98.2% for the rpoB/C-PCR, as well as 12.7% and 99.8% for the gyrA-PCR, respectively. The calculated A. butzleri prevalence within the assessed Ghanaian population was 14.7%. As indicated by test results obtained with high-titer spiked samples, cross-reactions of the hsp60-assay and rpoB/C-assay with phylogenetically related species such as A. cryaerophilus can occur but are less likely with phylogenetically more distant species like, e.g., A. lanthieri. In conclusion, the rpoB/C-assay showed the most promising performance characteristics as the only assay with sensitivity >95%, albeit associated with a broad 95%-confidence interval. In addition, this assay showed still-acceptable specificity of >98% in spite of the known cross-reactivity with phylogenetically closely related species such as A. cryaerophilus. If higher certainty is desired, the gyrA-assay with specificity close to 100% can be applied for confirmation testing with samples showing positive rpoB/C-PCR results. However, in case of a negative result in the gyrA-assay, this cannot reliably exclude the detection of A. butzleri in the rpoB/C-assay due to the gyrA-assay's very low sensitivity.

Comparison of the Anti-SARS-CoV-2 Surrogate Neutralization Assays by TECOmedical and DiaPROPH-Med with Samples from Vaccinated and Infected Individuals.

Anti-SARS-CoV-2-specific serological responses are a topic of ongoing evaluation studies. In the study presented here, the anti-SARS-CoV-2 surrogate neutralization assays by TECOmedical and DiaPROPH -Med were assessed in a head-to-head comparison with serum samples of individuals after vaccination as well as after previous infection with SARS-CoV-2. In case of discordant results, a cell culture-based neutralization assay was applied as a reference standard. The TECOmedical assay showed sensitivity and specificity of 100% and 61.3%, respectively, the DiaPROPH-Med assay 95.0% and 48.4%, respectively. As a side finding of the study, differences in the likelihood of expressing neutralizing antibodies could be shown for different exposition types. So, 60 of 81 (74.07%) of the samples with only one vaccination showed an expression of neutralizing antibodies in contrast to 85.71% (60 of 70 samples) of the samples with two vaccinations and 100% (40 of 40) of the samples from previously infected individuals. In conclusion, the both assays showed results similar to previous assessments. While the measured diagnostic accuracy of both assays requires further technical improvement of this diagnostic approach, as the calculated specificity values of 61.3% and 48.4%, respectively, appear acceptable for diagnostic use only in populations with a high percentage of positive subjects, but not at expectedly low positivity rates.

Case Report: Interferon- γ Rescues Monocytic Human Leukocyte Antigen Receptor (mHLA-DR) Function in a COVID-19 Patient With ARDS and Superinfection With Multiple MDR 4MRGN Bacterial Strains.

Background: CD14+ monocytes present antigens to adaptive immune cells via monocytic human leukocyte antigen receptor (mHLA-DR), which is described as an immunological synapse. Reduced levels of mHLA-DR can display an acquired immune defect, which is often found in sepsis and predisposes for secondary infections and fatal outcomes. Monocytic HLA-DR expression is reliably induced by interferon- γ (IFNγ) therapy. Case Report: We report a case of multidrug-resistant superinfected COVID-19 acute respiratory distress syndrome (ARDS) on extracorporeal membrane oxygenation (ECMO) support. The resistance profiles of the detected Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii and Citrobacter freundii isolates were equipped with resistance to all four antibiotic classes including carbapenems (4MRGN) and Cefiderocol in the case of K. pneumoniae. A causal therapeutic antibiotic strategy was not available. Therefore, we measured the immune status of the patient aiming to identify a potential acquired immune deficiency. Monocyte HLA-DR expression identified by FACS analysis revealed an expression level of 34% positive monocytes and suggested severe immunosuppression. We indicated IFNγ therapy, which resulted in a rapid increase in mHLA-DR expression (96%), rapid resolution of invasive bloodstream infection, and discharge from the hospital on day 70. Discussion: Superinfection is a dangerous complication of COVID-19 pneumonia, and sepsis-induced immunosuppression is a risk factor for it. Immunosuppression is expressed by a disturbed antigen presentation of monocytes to cells of the adaptive immune system. The case presented here is remarkable as no validated antibiotic regimen existed against the detected bacterial pathogens causing bloodstream infection and severe pneumonia in a patient suffering from COVID-19 ARDS. Possible restoration of the patient's own immunity by IFNγ was a plausible option to boost the patient's immune system, eliminate the identified 4MRGNs, and allow for lung recovery. This led to the conclusion that immune status monitoring is useful in complicated COVID-19-ARDS and that concomitant IFNγ therapy may support antibiotic strategies. Conclusion: After a compromised immune system has been detected by suppressed mHLA-DR levels, the immune system can be safely reactivated by IFNγ.

Risk Assessment for Molds in the Vicinity of a Child Requiring Peritoneal Dialysis Living in a Rural Northern German Area.

As well as severe immunosuppression, other predisposing factors may facilitate invasive mycosis caused by molds. Chronic kidney disease and the resulting peritoneal dialysis have been reported as factors putting patients at risk of fungal infections from environmental sources. We describe an environmental investigation undertaken to guide exposure prevention for a peritoneal dialysis patient with transient colonization of her nostrils by Lichtheimia corymbifera in a rural area of northern Germany. Systematic screening for airborne and surface-deposited molds enabled targeted recommendations to be made, although Lichtheimia corymbifera itself was not grown from the collected environmental samples. This communication is intended to illustrate how such an investigation can be performed on the basis of the environmental distribution of the molds and how preventive recommendations can be derived from the results.

Diagnosing SARS-CoV-2 with Antigen Testing, Transcription-Mediated Amplification and Real-Time PCR.

This study was performed as a head-to-head comparison of the performance characteristics of (1) two SARS-CoV-2-specific rapid antigen assays with real-time PCR as gold standard as well as (2) a fully automated high-throughput transcription-mediated amplification (TMA) assay and real-time PCR in a latent class analysis-based test comparison without a gold standard with several hundred samples in a low prevalence "real world" setting. Recorded sensitivity and specificity of the NADAL and the LumiraDx antigen assays and the Hologic Aptima SARS-CoV-2 TMA assay were 0.1429 (0.0194, 0.5835), 0.7644 (0.7016, 0.8174), and 0.7157 (0, 1) as well as 0.4545 (0.2022, 0.7326), 0.9954 (0.9817, 0.9988), and 0.9997 (not estimable), respectively. Agreement kappa between the positive results of the two antigen-based assays was 0.060 (0.002, 0.167) and 0.659 (0.492, 0.825) for TMA and real-time PCR. Samples with low viral load as indicated by cycle threshold (Ct) values > 30 were generally missed by both antigen assays, while 1:10 pooling suggested higher sensitivity of TMA compared to real-time PCR. In conclusion, both sensitivity and specificity speak in favor of the use of the LumiraDx rather than the NADAL antigen assay, while TMA results are comparably as accurate as PCR, when applied in a low prevalence setting.

Comparison of Self-Reported Sexual Activity Among Heterosexuals with Sexual Spread of Poorly Transmittable Agents: A Minimalistic Approach to Estimating Sexual Activity Based on HIV Incidence.

The aim of this study was to assess whether epidemics of sexually transmitted infections caused by poorly transmittable agents corresponded to self-reported sexual activity in a distinct population. To exemplify this, a model was used to investigate whether HIV infection incidences corresponded to the extent of sexual activity as assessed by a questionnaire-based study. The model suggested between 97 and 486 sexual contacts per German individual during a sexually active lifetime based on the annual HIV incidence of 680 among the heterosexual population reported by the German National Health Authority. This is in line with the estimated 296 sexual contacts during one's lifetime, which was indicated by questionnaire respondents. The model confirms the correspondence of self-reported sexual activity with HIV incidence as reported by the German National Health Authority. Accordingly, HIV incidence- and prevalence-based modeling of sexual activity in a population provides crude estimations in situations where a range of uncertainty is acceptable. The model's veracity is limited by a number of assumptions necessitated by the paucity of data. Nevertheless, the model may be suitable in settings where severe reporting bias has to be expected for legal or socio-cultural reasons.

On detection thresholds-a review on diagnostic approaches in the infectious disease laboratory and the interpretation of their results.

Diagnostic testing in the infectious disease laboratory facilitates decision-making by physicians at the bedside as well as epidemiological assessments and surveillance at study level. Problems may arise if test results are uncritically considered as being the same as the unknown true value. To allow a better understanding, the influence of external factors on the interpretation of test results is introduced with the example of prevalence, followed by the presentation of strengths and weaknesses of important techniques in the infectious disease laboratory like microscopy, cultural diagnostics, serology, mass spectrometry, nucleic acid amplification and hypothesis-free metagenomic sequencing with focus on basic, high-technology and potential future approaches. Special problems like multiplex testing as well as uncertainty of test evaluations, if no gold standard is available, are also stressed with a final glimpse on emerging future technologies for the infectious disease laboratory. In the conclusions, suitability for point-of-care-testing and field laboratory applications is summarized. The aim is to illustrate the limitations of diagnostic accuracy to both clinicians and study planners and to stress the importance of close cooperation with experts in laboratory disciplines so as to avoid potentially critical misunderstandings due to inappropriate interpretation of diagnostic test results.

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.

Pharmaceutical interactions between antiretroviral and antimalarial drugs used in chemoprophylaxis.

Human immunodeficiency virus (HIV) is the causative agent of the Acquired Immunodeficiency Syndrome (AIDS). The pandemic is believed to have originated within the Northern Congo basin covering large parts of the Democratic Republic of Congo, the Republic of Congo, the Central African Republic, Cameroon and Gabon. Although over decades, HIV-1 has spread throughout the World leaving no country unaffected, sub-Saharan Africa remains the region with more than 80% of all infected individuals. The HIV-2 epidemic has largely remained restricted to West Africa along the Upper Guinean forests. Co-incident with these regions of highest HIV distribution is a part of the malaria belt and therefore, co-infections are common. In this review we carve out the consequences of HIV transmission prevention and synchronous malaria prophylaxis during occupational or leisure travelling activities within this World region. In particular, we elaborate on considering pre-existing drug resistances of both, the malaria parasites and the immunodeficiency viruses, when determining a combination for prophylactic and, if necessary, post-expositional measures with a focus on the compatibility of both medications.

Fluorescence in situ hybridization (FISH) in the microbiological diagnostic routine laboratory: a review.

Early identification of microbial pathogens is essential for rational and conservative antibiotic use especially in the case of known regional resistance patterns. Here, we describe fluorescence in situ hybridization (FISH) as one of the rapid methods for easy identification of microbial pathogens, and its advantages and disadvantages for the diagnosis of pathogens in human infections in the laboratory diagnostic routine. Binding of short fluorescence-labeled DNA or nucleic acid-mimicking PNA probes to ribosomes of infectious agents with consecutive analysis by fluorescence microscopy allows identification of bacterial and eukaryotic pathogens at genus or species level. FISH analysis leads to immediate differentiation of infectious agents without delay due to the need for microbial culture. As a microscopic technique, FISH has the unique potential to provide information about spatial resolution, morphology and identification of key pathogens in mixed species samples. On-going automation and commercialization of the FISH procedure has led to significant shortening of the time-to-result and increased test reliability. FISH is a useful tool for the rapid initial identification of microbial pathogens, even from primary materials. Among the rapidly developing alternative techniques, FISH serves as a bridging technology between microscopy, microbial culture, biochemical identification and molecular diagnostic procedures.

Microbiological laboratory diagnostics of neglected zoonotic diseases (NZDs).

This review reports on laboratory diagnostic approaches for selected, highly pathogenic neglected zoonotic diseases, i.e. anthrax, bovine tuberculosis, brucellosis, echinococcosis, leishmaniasis, rabies, Taenia solium-associated diseases (neuro-/cysticercosis & taeniasis) and trypanosomiasis. Diagnostic options, including microscopy, culture, matrix-assisted laser-desorption-ionisation time-of-flight mass spectrometry, molecular approaches and serology are introduced. These procedures are critically discussed regarding their diagnostic reliability and state of evaluation. For rare diseases reliable evaluation data are scarce due to the rarity of samples. If bio-safety level 3 is required for cultural growth, but such high standards of laboratory infrastructure are not available, serological and molecular approaches from inactivated sample material might be alternatives. Multiple subsequent testing using various test platforms in a stepwise approach may improve sensitivity and specificity. Cheap and easy to use tests, usually called "rapid diagnostic tests" (RDTs) may impact disease control measures, but should not preclude developing countries from state of the art diagnostics.

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.

Towards proteomic species barcoding of fungi - An example using Scedosporium/Pseudallescheria complex isolates.

MALDI-ToF mass spectrometry offers fast and reliable species identification for bacteria and yeasts under clinical routine conditions. Here, we produced mass spectra for identification of clinically important species of the Pseudallescheria/Scedosporium complex using the recently suggested new nomenclature and use this example to discuss to what extent the principle of DNA barcoding might be transferred to mass spectrometry.

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.

Difficult identification of Haemophilus influenzae, a typical cause of upper respiratory tract infections, in the microbiological diagnostic routine.

Haemophilus influenzae is a key pathogen of upper respiratory tract infections. Its reliable discrimination from nonpathogenic Haemophilus spp. is necessary because merely colonizing bacteria are frequent at primarily unsterile sites. Due to close phylogenetic relationship, it is not easy to discriminate H. influenzae from the colonizer Haemophilus haemolyticus. The frequency of H. haemolyticus isolations depends on factors like sampling site, patient condition, and geographic region. Biochemical discrimination has been shown to be nonreliable. Multiplex PCR including marker genes like sodC, fucK, and hpd or sequencing of the 16S rRNA gene, the P6 gene, or multilocus-sequence-typing is more promising. For the diagnostic routine, such techniques are too expensive and laborious. If available, matrix-assisted laser-desorption-ionization time-of-flight mass spectrometry is a routine-compatible option and should be used in the first line. However, the used database should contain well-defined reference spectra, and the spectral difference between H. influenzae and H. haemolyticus is small. Fluorescence in-situ hybridization is an option for less well-equipped laboratories, but the available protocol will not lead to conclusive results in all instances. It can be used as a second line approach. Occasional ambiguous results have to be resolved by alternative molecular methods like 16S rRNA gene sequencing.

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.

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.

Effects of easy-to-perform procedures to reduce bacterial colonization with Streptococcus mutans and Staphylococcus aureus on toothbrushes.

It is well known that dental caries and periodontitis are the consequence of bacterial colonization and biofilm formation on the enamel surface. The continuous presence of bacterial biofilms on the tooth surface results in demineralization of the tooth enamel and induces an inflammatory reaction of the surrounding gums (gingivitis). The retention and survival of microorganisms on toothbrushes pose a threat of recontamination especially for certain patients at risk for systemic infections originating from the oral cavity, e.g., after T-cell depleted bone marrow transplantation. Thus, the effects of different decolonization schemes on bacterial colonization of toothbrushes were analyzed, in order to demonstrate their applicability to reduce the likelihood of (auto-)reinfections. Toothbrushes were intentionally contaminated with standardized suspensions of Streptococcus mutans or Staphylococcus aureus. Afterwards, the toothbrushes were exposed to rinsing under distilled water, rinsing and drying for 24 h, 0.2% chlorhexidine-based decolonization, or ultraviolet (UV) radiation. The remaining colony forming units were compared with freshly contaminated positive controls. Each experiment was nine-fold repeated. Bi-factorial variance analysis was performed; significance was accepted at P < 0.05. All tested procedures led to a significant reduction of bacteral colonization irrespective of the toothbrush model, the brush head type, or the acitivity state. Chlorhexidine-based decolonization was shown to be superior to rinsing and slightly superior to rinsing and drying for 24 h, while UV radiation was similarly effective as chlorhexidine. UV radiation was slightly less prone to species-dependent limitations of its decolonizing effects by bristle thickness of toothbrushes than chlorhexidin. Reduction of bacterial colonization of toothbrushes might reduce the risk of maintaining bacterial infections of the upper respiratory tract. Accordingly, respective procedures are advisable, particularly as they are cheap and easy to perform.