Diversity of Staphylococcus aureus isolated from nares of ruminants.

AIMS: To examine the diversity of Staphylococcus aureus isolated from nasal swabs of ruminants in Rwanda. METHODS AND RESULTS: A total of 454 nasal swabs from 203 cows, 170 goats, and 81 sheep were examined for the presence of S. aureus, and 30 S. aureus isolates were detected and characterized pheno- and genotypically. Resistance to penicillin and/or tetracycline was observed. The isolates were assigned to eight different spa types (t21057 (novel), t10103, t18853, t20842, t318, t355, t458, and t9432) belonging to six clonal complexes (CCs) (CC152, CC30, CC3591, CC3666, CC522, and CC97). Panton-Valentine leukocidin (PVL) genes (lukF-PV/lukS-PV), the bovine leukocidin genes (lukM/lukF-P83), and the human and bovine variants of the toxic shock syndrome toxin gene tst-1 variants were detected. CONCLUSION: These findings demonstrate that the nares of ruminants in Rwanda are colonized with mastitis-associated S. aureus, including lineages that are also carried by humans, underscoring the zoonotic risk, especially for livestock keepers. These results highlight the crucial importance of hygiene measures when handling livestock.

Antimicrobial Ionic Liquids: Ante-Mortem Mechanisms of Pathogenic EPEC and MRSA Examined by FTIR Spectroscopy.

Ionic liquids (ILs) have gained considerable attention due to their versatile and designable properties. ILs show great potential as antibacterial agents, but understanding the mechanism of attack on bacterial cells is essential to ensure the optimal design of IL-based biocides. The final aim is to achieve maximum efficacy while minimising toxicity and preventing resistance development in target organisms. In this study, we examined a dose-response analysis of ILs' antimicrobial activity against two pathogenic bacteria with different Gram types in terms of molecular responses on a cellular level using Fourier-transform infrared (FTIR) spectroscopy. In total, 18 ILs with different antimicrobial active motifs were evaluated on the Gram-negative enteropathogenic Escherichia coli (EPEC) and Gram-positive methicillin-resistant Staphylococcus aureus (MRSA). The results showed that most ILs impact bacterial proteins with increasing concentration but have a minimal effect on cellular membranes. Dose-response spectral analysis revealed a distinct ante-mortem response against certain ILs for MRSA but not for EPEC. We found that at sub-lethal concentrations, MRSA actively changed their membrane composition to counteract the damaging effect induced by the ILs. This suggests a new adaptive mechanism of Gram-positive bacteria against ILs and demonstrates the need for a better understanding before using such substances as novel antimicrobials.

Proteomic and immunoproteomic insights into the exoproteome of Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia.

Porcine pleuropneumonia caused by Actinobacillus pleuropneumoniae affects pig health status and the swine industry worldwide. Despite the extensive number of studies focused on A. pleuropneumoniae infection and vaccine development, a thorough analysis of the A. pleuropneumoniae exoproteome is still missing. Using a complementary approach of quantitative proteomics and immunoproteomics we gained an in-depth insight into the A. pleuropneumoniae serotype 2 exoproteome, which provides the basis for future functional studies. Label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed 593 exoproteins, of which 104 were predicted to be virulence factors. The RTX toxins ApxIIA and ApxIIIA -were found to be the most abundant proteins in the A. pleuropneumoniae serotype 2 exoproteome. Furthermore, the ApxIVA toxin was one of the proteins showing the highest abundance, although ApxIVA is commonly assumed to be expressed exclusively in vivo. Our study revealed several antigens, including proteins with moonlight functions, such as the elongation factor (EF)-Tu, and proteins linked to specific metabolic traits, such as the maltodextrin-binding protein MalE, that warrant future functional characterization and might present potential targets for novel therapeutics and vaccines. Our Ig-classes specific serological proteome analysis (SERPA) approach allowed us to explore the development of the host humoral immune response over the course of the infection. These SERPAs pinpointed proteins that might play a key role in virulence and persistence and showed that the immune response to the different Apx toxins is distinct. For instance, our results indicate that the ApxIIIA toxin has properties of a thymus-independent antigen, which should be studied in more detail.

Within-Host Adaptation of Staphylococcus aureus in a Bovine Mastitis Infection Is Associated with Increased Cytotoxicity.

Within-host adaptation is a typical feature of chronic, persistent Staphylococcus aureus infections. Research projects addressing adaptive changes due to bacterial in-host evolution increase our understanding of the pathogen's strategies to survive and persist for a long time in various hosts such as human and bovine. In this study, we investigated the adaptive processes of S. aureus during chronic, persistent bovine mastitis using a previously isolated isogenic strain pair from a dairy cow with chronic, subclinical mastitis, in which the last variant (host-adapted, Sigma factor SigB-deficient) quickly replaced the initial, dominant variant. The strain pair was cultivated under specific in vitro infection-relevant growth-limiting conditions (iron-depleted RPMI under oxygen limitation). We used a combinatory approach of surfaceomics, molecular spectroscopic fingerprinting and in vitro phenotypic assays. Cellular cytotoxicity assays using red blood cells and bovine mammary epithelial cells (MAC-T) revealed changes towards a more cytotoxic phenotype in the host-adapted isolate with an increased alpha-hemolysin (α-toxin) secretion, suggesting an improved capacity to penetrate and disseminate the udder tissue. Our results foster the hypothesis that within-host evolved SigB-deficiency favours extracellular persistence in S. aureus infections. Here, we provide new insights into one possible adaptive strategy employed by S. aureus during chronic, bovine mastitis, and we emphasise the need to analyse genotype-phenotype associations under different infection-relevant growth conditions.

Host-pathogen interplay at primary infection sites in pigs challenged with Actinobacillus pleuropneumoniae.

BACKGROUND: Actinobacillus (A.) pleuropneumoniae is the causative agent of porcine pleuropneumonia and causes significant losses in the pig industry worldwide. Early host immune response is crucial for further progression of the disease. A. pleuropneumoniae is either rapidly eliminated by the immune system or switches to a long-term persistent form. To gain insight into the host-pathogen interaction during the early stages of infection, pigs were inoculated intratracheally with A. pleuropneumoniae serotype 2 and humanely euthanized eight hours after infection. Gene expression studies of inflammatory cytokines and the acute phase proteins haptoglobin, serum amyloid A and C-reactive protein were carried out by RT-qPCR from the lung, liver, tonsils and salivary gland. In addition, the concentration of cytokines and acute phase proteins were measured by quantitative immunoassays in bronchoalveolar lavage fluid, serum and saliva. In parallel to the analyses of host response, the impact of the host on the bacterial pathogen was assessed on a metabolic level. For the latter, Fourier-Transform Infrared (FTIR-) spectroscopy was employed. RESULTS: Significant cytokine and acute phase protein gene expression was detected in the lung and the salivary gland however this was not observed in the tonsils. In parallel to the analyses of host response, the impact of the host on the bacterial pathogen was assessed on a metabolic level. For the latter investigations, Fourier-Transform Infrared (FTIR-) spectroscopy was employed. The bacteria isolated from the upper and lower respiratory tract showed distinct IR spectral patterns reflecting the organ-specific acute phase response of the host. CONCLUSIONS: In summary, this study implies a metabolic adaptation of A. pleuropneumoniae to the porcine upper respiratory tract already during early infection, which might indicate a first step towards the persistence of A. pleuropneumoniae. Not only in lung, but also in the salivary gland an increased inflammatory gene expression was detectable during the acute stage of infection.

Carriage of meticillin-resistant staphylococci between humans and animals on a small farm.

BACKGROUND: Meticillin-resistant staphylococci (MRS) are pathogens of increasing importance to human and animal health worldwide. Transmission of meticillin-resistant Staphylococcus aureus (MRSA) between animals and humans has been well documented. By contrast, information about transmission of other Staphylococcus spp. is limited. HYPOTHESIS/OBJECTIVES: The aim of this study was to screen animals and humans on a small farm for nasal carriage of MRS and to assess interspecies exchange. METHODS: After detection of MRSA in a lung sample of a deceased cat, which lived on a small mixed farm, nasal swabs were taken within two weeks, four and 16 months from other animals of various species and humans living on the farm. Swabs were cultured for MRS which were then characterized molecularly. RESULTS: MRSA and meticillin-resistant coagulase negative staphylococci (MRCoNS), including Staphylococcus haemolyticus, S. epidermidis and S. fleurettii, were isolated from humans and different animal species. Typing of the MRS revealed isolates with the same characteristics in different human and animal hosts. CONCLUSIONS AND CLINICAL IMPORTANCE: To the best of the authors' knowledge, this is the first report of carriage of both MRSA and MRCoNS among humans and various animals within a shared environment. The detection of strains with indistinguishable molecular characteristics strongly suggested transmission of these MRS between the various animal species and humans.

Diversity of Staphylococcus aureus associated with mastitis from dairy cows in Rwanda.

OBJECTIVES: The objective of the present study was to examine the diversity of Staphylococcus aureus from mastitis milk samples of cows in Rwanda. METHODS: A total of 1080 quarter milk samples from 279 dairy cows were collected in 80 different farms from all five provinces of Rwanda. In total, 135 S. aureus isolates were obtained and subjected to genotyping (spa typing, DNA microarray, whole-genome sequencing (WGS)), antimicrobial susceptibility testing (AST) and phenotypic profiling by Fourier Transform Infrared (FTIR) spectroscopy (including capsular serotyping). RESULTS: Resistance to penicillin and/or tetracycline was most frequently observed. Ten sequence types (STs) (ST1, ST151, ST152, ST5477, ST700, ST7110, ST7983, ST7984, ST8320, ST97) belonging to seven clonal complexes (CCs) (CC1, CC130, CC152, CC3591, CC3666, CC705, CC97) were detected. The Panton-Valentine leukocidin (PVL) genes (lukF-PV/lukS-PV), the bovine leukocidin genes (lukM/lukF-P83) and the human and bovine toxic shock syndrome toxin gene tst-1 variants were detected. FTIR-based capsular serotyping showed CC-specific differences. Most CC97 (cap5 allele) isolates were primarily nonencapsulated (82%), whereas isolates of CC3591 and CC3666 (cap8 allele) were mostly encapsulated (86.4% and 57.8%, respectively). Our results underline the widespread global distribution of cattle-adapted CC97. CONCLUSION: The presence of CC3591 and CC3666 in bovine mastitis suggests an important role in cattle health and dairy production in Rwanda. The results of the present study support the need for a rigorous One-Health Surveillance program of the bovine-human interface.

Rapid and reliable identification of Staphylococcus aureus capsular serotypes by means of artificial neural network-assisted Fourier transform infrared spectroscopy.

Staphylococcus aureus capsular polysaccharides (CP) are important virulence factors and represent putative targets for vaccine development. Therefore, the purpose of this study was to develop a high-throughput method to identify and discriminate the clinically important S. aureus capsular serotypes 5, 8, and NT (nontypeable). A comprehensive set of clinical isolates derived from different origins and control strains, representative for each serotype, were used to establish a CP typing system based on Fourier transform infrared (FTIR) spectroscopy and chemometric techniques. By combining FTIR spectroscopy with artificial neuronal network (ANN) analysis, a system was successfully established, allowing a rapid identification and discrimination of all three serotypes. The overall accuracy of the ANN-assisted FTIR spectroscopy CP typing system was 96.7% for the internal validation and 98.2% for the external validation. One isolate in the internal validation and one isolate in the external validation failed in the classification procedure, but none of the isolates was incorrectly classified. The present study demonstrates that ANN-assisted FTIR spectroscopy allows a rapid and reliable discrimination of S. aureus capsular serotypes. It is suitable for diagnostic as well as large-scale epidemiologic surveillance of S. aureus capsule expression and provides useful information with respect to chronicity of infection.

Prevalence of the SigB-Deficient Phenotype among Clinical Staphylococcus aureus Isolates Linked to Bovine Mastitis.

Phenotypic adaptation has been associated with persistent, therapy-resistant Staphylococcus aureus infections. Recently, we described within-host evolution towards a Sigma factor B (SigB)-deficient phenotype in a non-human host, a naturally infected dairy cow with chronic, persistent mastitis. However, to our knowledge, the prevalence of SigB deficiency among clinical S. aureus isolates remains unknown. In this study, we screened a collection of bovine mastitis isolates for phenotypic traits typical for SigB deficiency: decreased carotenoid pigmentation, increased proteolysis, secretion of α-hemolysin and exoproteins. Overall, 8 out of 77 (10.4%) isolates of our bovine mastitis collection exhibited the SigB-deficient phenotype. These isolates were assigned to various clonal complexes (CC8, CC9, CC97, CC151, CC3666). We further demonstrated a strong positive correlation between asp23-expression (a marker of SigB activity) and carotenoid pigmentation (r = 0.6359, p = 0.0008), underlining the role of pigmentation as a valuable predictor of the functional status of SigB. Sequencing of the sigB operon (mazEF-rsbUVW-sigB) indicated the phosphatase domain of the RsbU protein as a primary target of mutations leading to SigB deficiency. Indeed, by exchanging single nucleotides in rsbU, we could either induce SigB deficiency or restore the SigB phenotype, demonstrating the pivotal role of RsbU for SigB functionality. The data presented highlight the clinical relevance of SigB deficiency, and future studies are needed to exploit its role in staphylococcal infections.

Cross-species communication via agr controls phage susceptibility in Staphylococcus aureus.

Bacteria use quorum sensing (QS) to coordinate group behavior in response to cell density, and some bacterial viruses (phages) also respond to QS. In Staphylococcus aureus, the agr-encoded QS system relies on accumulation of auto-inducing cyclic peptides (AIPs). Other staphylococci also produce AIPs of which many inhibit S. aureus agr. We show that agr induction reduces expression of tarM, encoding a glycosyltransferase responsible for α-N-acetylglucosamine modification of the major S. aureus phage receptor, the wall teichoic acids. This allows lytic phage Stab20 and related phages to infect and kill S. aureus. However, in mixed communities, producers of inhibitory AIPs like S. haemolyticus, S. caprae, and S. pseudintermedius inhibit S. aureus agr, thereby impeding phage infection. Our results demonstrate that cross-species interactions dramatically impact phage susceptibility. These interactions likely influence microbial ecology and impact the efficacy of phages in medical and biotechnological applications such as phage therapy.

Tyrosine kinase 2 controls IL-1ß production at the translational level.

IL-1beta is an important proinflammatory cytokine with a major role in several inflammatory diseases. Expression of IL-1beta is tightly regulated at the level of transcription, mRNA stability, and proteolytic processing. In this study, we report that IL-1beta expression in response to LPS is also regulated at the translational level. LPS-induced IL-1beta protein levels in macrophages derived from murine bone marrow are markedly increased in the absence of tyrosine kinase 2 (Tyk2). Increased IL-1beta is found intra- and extracellularly, irrespective of the efficiency of IL-1beta processing. We show that the absence of Tyk2 results both in higher translational rates and in enhanced association of IL-1beta mRNA with polysomes. Induction and stability of IL-1beta mRNA are not affected by the lack of Tyk2. We show further that the Tyk2-dependent translational inhibition is mediated by autocrine/paracrine type I IFN signaling and requires signal transducer and activator of transcription 1. Enhanced IL-1beta production in Tyk2- and IFN receptor 1-deficient macrophages is also observed following Listeria monocytogenes infection. Taken together, the data describe a novel mechanism for the control of IL-1beta synthesis.

YjbH regulates virulence genes expression and oxidative stress resistance in Staphylococcus aureus.

We previously reported that disruption of the yjbI gene reduced virulence of Staphylococcus aureus. In this study, we found virulence in both silkworms and mice was restored by introducing the yjbH gene but not the yjbI gene to both yjbI and yjbH genes-disrupted mutants, suggesting that yjbH, the gene downstream to the yjbI gene in a two-gene operon-yjbIH, is responsible for this phenomenon. We further observed a decrease in various surface-associated proteins and changes in cell envelope glycostructures in the mutants. RNA-seq analysis revealed that disruption of the yjbI and the yjbH genes resulted in differential expression of a broad range of genes, notably, significant downregulation of genes involved in virulence and oxidative stress. Administration of N-acetyl-L-cysteine, a free-radical scavenger, restored the virulence in both the mutants. Our findings suggested that YjbH plays a role in staphylococcal pathogenicity by regulating virulence gene expression, affecting the bacterial surface structure, and conferring resistance to oxidative stress in a host.

Salicylic acid stabilizes Staphylococcus aureus biofilm by impairing the agr quorum-sensing system.

Salicylic acid (SAL) has recently been shown to induce biofilm formation in Staphylococcus aureus and to affect the expression of virulence factors. This study was aimed to investigate the effect of SAL on the regulatory agr system and its impact on S. aureus biofilm formation. The agr quorum-sensing system, which is a central regulator in S. aureus pathogenicity, plays a pivotal role in the dispersal of S. aureus mature biofilms and contributes to the creation of new colonization sites. Here, we demonstrate that SAL impairs biofilm dispersal by interfering with agr expression. As revealed by our work, protease and surfactant molecule production is diminished, and bacterial cell autolysis is also negatively affected by SAL. Furthermore, as a consequence of SAL treatment, the S. aureus biofilm matrix revealed the lack of extracellular DNA. In silico docking and simulation of molecular dynamics provided evidence for a potential interaction of AgrA and SAL, resulting in reduced activity of the agr system. In conclusion, SAL stabilized the mature S. aureus biofilms, which may prevent bacterial cell dissemination. However, it may foster the establishment of infections locally and consequently increase bacterial persistence leading to therapeutic failure.

The Novel Membrane-Associated Auxiliary Factors AuxA and AuxB Modulate ß-lactam Resistance in MRSA by stabilizing Lipoteichoic Acids.

A major determinant of ß-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA) is the drug insensitive transpeptidase, PBP2a, encoded by mecA. Full expression of the resistance phenotype requires auxiliary factors. Two such factors, auxiliary factor A (auxA, SAUSA300_0980) and B (auxB, SAUSA300_1003), were identified in a screen against mutants with increased susceptibility to ß-lactams in the MRSA strain, JE2. auxA and auxB encode transmembrane proteins, with AuxA predicted to be a transporter. Inactivation of auxA or auxB enhanced ß-lactam susceptibility in community-, hospital- and livestock-associated MRSA strains without affecting PBP2a expression, peptidoglycan cross-linking or wall teichoic acid synthesis. Both mutants displayed increased susceptibility to inhibitors of lipoteichoic acid (LTA) synthesis and alanylation pathways and released LTA even in the absence of ß-lactams. The ß-lactam susceptibility of the aux mutants was suppressed by mutations inactivating gdpP, which was previously found to allow growth of mutants lacking the lipoteichoic synthase enzyme, LtaS. Using the Galleria mellonella infection model, enhanced survival of larvae inoculated with either auxA or auxB mutants was observed compared with the wild-type strain following treatment with amoxicillin. These results indicate that AuxA and AuxB are central for LTA stability and potential inhibitors can be tools to re-sensitize MRSA strains to ß-lactams and combat MRSA infections.

Vibrational Spectroscopy of Peritoneal Dialysis Effluent for Rapid Assessment of Patient Characteristics.

Peritoneal dialysis (PD) offers specific advantages over hemodialysis, enabling increased autonomy of patients with end-stage renal disease, but PD-related complications need to be detected in a timely manner. Fourier transform infrared (FTIR) spectroscopy could provide rapid and essential insights into the patients' risk profiles via molecular fingerprinting of PD effluent, an abundant waste material that is rich in biological information. In this study, we measured FTIR spectroscopic profiles in PD effluent from patients taking part in a randomized controlled trial of alanyl-glutamine addition to the PD-fluid. Principal component analysis of FTIR spectra enabled us to differentiate between effluent samples from patients immediately after completion of instillation of the PD-fluid into the patients' cavity and 4 h later as well as between patients receiving PD-fluid supplemented with 8 mM alanyl-glutamine compared with control. Moreover, feasibility of FTIR spectroscopy coupled to supervised classification algorithms to predict patient-, PD-, as well as immune-associated parameters were investigated. PD modality (manual continuous ambulatory PD (CAPD) vs. cycler-assisted automated PD (APD)), residual urine output, ultrafiltration, transport parameters, and cytokine concentrations showed high predictive potential. This study provides proof-of-principle that molecular signatures determined by FTIR spectroscopy of PD effluent, combined with machine learning, are suitable for cost-effective, high-throughput diagnostic purposes in PD.

FTIR Spectroscopy Suggests a Revised Mode of Action for the Cationic Side-Chain Effect of Ionic Liquids.

Over the past decades, ionic liquids (ILs) have gained considerable attention from the scientific community because of their versatile and designable properties. As a result, there are numerous IL applications, not only in organic synthesis, catalysis, or extraction but also as active pharmaceutical ingredients or novel antimicrobials. While considerable effort has been put into developing quantitative structure-activity relationship (QSAR) models for IL toxicity prediction, little is known about their actual mode of action. In this study, Fourier transform infrared (FTIR) spectroscopy is used to monitor IL induced molecular responses directly at the cellular level. Investigation of the well-known cationic alkyl side-chain effect (increasing side-chain length leads to increasing toxicity) of imidazolium- and ammonium-based ILs on two bacterial pathogens, enteropathogenic  Escherichia coli (EPEC) and methicillin-resistant Staphylococcus aureus (MRSA), surprisingly revealed two distinct modes of action. Contrary to prior models, it was only for [TMC16A][Cl], where a molecular response in the membrane was found, while ILs with shorter side-chain lengths predominantly affected bacterial proteins. The results of this study highlight the importance of further direct investigations of the impact of ILs at the cellular level to improve toxicity prediction and assess the usefulness of spectroscopic methods, such as FTIR spectroscopy at achieving this goal.

Within-host evolution of bovine Staphylococcus aureus selects for a SigB-deficient pathotype characterized by reduced virulence but enhanced proteolytic activity and biofilm formation.

Staphylococcus aureus is a major cause of bovine mastitis, commonly leading to long-lasting, persistent and recurrent infections. Thereby, S. aureus constantly refines and permanently adapts to the bovine udder environment. In this work, we followed S. aureus within-host adaptation over the course of three months in a naturally infected dairy cattle with chronic, subclinical mastitis. Whole genome sequence analysis revealed a complete replacement of the initial predominant variant by another isogenic variant. We report for the first time within-host evolution towards a sigma factor SigB-deficient pathotype in S. aureus bovine mastitis, associated with a single nucleotide polymorphism in rsbU (G368A → G122D), a contributor to SigB-functionality. The emerged SigB-deficient pathotype exhibits a substantial shift to new phenotypic traits comprising strong proteolytic activity and poly-N-acetylglucosamine (PNAG)-based biofilm production. This possibly unlocks new nutritional resources and promotes immune evasion, presumably facilitating extracellular persistence within the host. Moreover, we observed an adaptation towards attenuated virulence using a mouse infection model. This study extends the role of sigma factor SigB in S. aureus pathogenesis, so far described to be required for intracellular persistence during chronic infections. Our findings suggest that S. aureus SigB-deficiency is an alternative mechanism for persistence and underpin the clinical relevance of staphylococcal SigB-deficient variants which are consistently isolated during human chronic infections.

Characterization of Antibiotic and Biocide Resistance Genes and Virulence Factors of Staphylococcus Species Associated with Bovine Mastitis in Rwanda.

The present study was conducted from July to August 2018 on milk samples taken at dairy farms in the Northern Province and Kigali District of Rwanda in order to identify Staphylococcus spp. associated with bovine intramammary infection. A total of 161 staphylococcal isolates originating from quarter milk samples of 112 crossbred dairy cattle were included in the study. Antimicrobial susceptibility testing was performed and isolates were examined for the presence of various resistance genes. Staphylococcus aureus isolates were also analyzed for the presence of virulence factors, genotyped by spa typing and further phenotypically subtyped for capsule expression using Fourier Transform Infrared (FTIR) spectroscopy. Selected S. aureus were characterized using DNA microarray technology, multi-locus sequence typing (MLST) and whole-genome sequencing. All mecA-positive staphylococci were further genotyped using dru typing. In total, 14 different staphylococcal species were detected, with S. aureus being most prevalent (26.7%), followed by S. xylosus (22.4%) and S. haemolyticus (14.9%). A high number of isolates was resistant to penicillin and tetracycline. Various antimicrobial and biocide resistance genes were detected. Among S. aureus, the Panton-Valentine leukocidin (PVL) genes, as well as bovine leukocidin (LukM/LukF-P83) genes, were detected in two and three isolates, respectively, of which two also carried the toxic shock syndrome toxin gene tsst-1 bovine variant. t1236 was the predominant spa type. FTIR-based capsule serotyping revealed a high prevalence of non-encapsulated S. aureus isolates (89.5%). The majority of the selected S. aureus isolates belonged to clonal complex (CC) 97 which was determined using DNA microarray based assignment. Three new MLST sequence types were detected.

The impact of tyrosine kinase 2 (Tyk2) on the proteome of murine macrophages and their response to lipopolysaccharide (LPS).

Tyrosine kinase 2 (Tyk2) belongs to the Janus kinase (Jak) family and is involved in signalling via a number of cytokines. Tyk2-deficient mice are highly resistant to lipopolysaccharide (LPS)-induced endotoxin shock. Macrophages are key players in the pathogenesis of endotoxin shock and, accordingly, defects in the LPS responses of Tyk2(-/-) macrophages have been reported. In the present study, the molecular role of Tyk2 is investigated in more detail using a proteomics approach. 2-D DIGE was applied to compare protein patterns from wild-type and Tyk2(-/-) macrophages and revealed significant differences in protein expression patterns between the genotypes before and after LPS treatment. Twenty-one proteins deriving from 25 differentially expressed spots were identified by MALDI/ESI MS. Among them, we show for N-myc interactor that its mRNA transcription/stability is positively influenced by Tyk2. In contrast, LPS-induced expression of plasminogen activator 2 protein but not mRNA is strongly enhanced in the absence of Tyk2. Our data furthermore suggest an influence of Tyk2 on the subcellular distribution of elongation factor 2 and on LPS-mediated changes in the peroxiredoxin 1 spot pattern. Thus, our results imply regulatory roles of Tyk2 at multiple levels and establish novel connections between Tyk2 and several cellular proteins.

Distinct phenotypic traits of Staphylococcus aureus are associated with persistent, contagious bovine intramammary infections.

Staphylococcus aureus causing persistent, recurrent bovine intramammary infections are still a major challenge to dairy farming. Generally, one or a few clonal lineages are predominant in dairy herds, indicating animal-to-animal transfers and the existence of distinct pathotypic traits. The aim of this study was to determine if long term persistence and spreading of S. aureus are associated with specific phenotypic traits, including cellular invasion, cytotoxicity and biofilm formation. Mastitis isolates were collected over a 3-years period from a single dairy herd, resulting in two persistent subtypes, the high within-herd prevalent subtype ST9 (CC9)-methicillin-susceptible S. aureus (MSSA), designated HP/ST9, and the low within-herd prevalent subtype ST504 (CC705)-MSSA, designated LP/ST504. Characterization of the two different coexisting persistent subtypes showed that the following phenotypic traits are particularly associated with high within-herd prevalence: lack of capsular polysaccharide expression, high cellular invasiveness, low cytotoxicity and high biofilm/ poly-N-acetylglucosamine (PNAG) production, which may concomitantly contribute to the spreading of HP/ST9 within the herd. By contrast to HP/ST9, LP/ST504 is characterized by the formation of colony dendrites, which may help the bacteria to access deeper tissues as niches for persistence in single animals. Thus, within a single herd, two different types of persistence can be found in parallel, allowing longtime persistence of S. aureus in dairy cattle. Furthermore, this study indicates that ST9 (CC9)-MSSA strains, which are currently thought to have their primary reservoir in swine and humans, can also successfully spread to new hosts and persist in dairy herds for years.