Methicillin-resistant Staphylococcus aureus alters cell wall glycosylation to evade immunity.

Methicillin-resistant Staphylococcus aureus (MRSA) is a frequent cause of difficult-to-treat, often fatal infections in humans1,2. Most humans have antibodies against S. aureus, but these are highly variable and often not protective in immunocompromised patients3. Previous vaccine development programs have not been successful4. A large percentage of human antibodies against S. aureus target wall teichoic acid (WTA), a ribitol-phosphate (RboP) surface polymer modified with N-acetylglucosamine (GlcNAc)5,6. It is currently unknown whether the immune evasion capacities of MRSA are due to variation of dominant surface epitopes such as those associated with WTA. Here we show that a considerable proportion of the prominent healthcare-associated and livestock-associated MRSA clones CC5 and CC398, respectively, contain prophages that encode an alternative WTA glycosyltransferase. This enzyme, TarP, transfers GlcNAc to a different hydroxyl group of the WTA RboP than the standard enzyme TarS7, with important consequences for immune recognition. TarP-glycosylated WTA elicits 7.5-40-fold lower levels of immunoglobulin G in mice than TarS-modified WTA. Consistent with this, human sera contained only low levels of antibodies against TarP-modified WTA. Notably, mice immunized with TarS-modified WTA were not protected against infection with tarP-expressing MRSA, indicating that TarP is crucial for the capacity of S. aureus to evade host defences. High-resolution structural analyses of TarP bound to WTA components and uridine diphosphate GlcNAc (UDP-GlcNAc) explain the mechanism of altered RboP glycosylation and form a template for targeted inhibition of TarP. Our study reveals an immune evasion strategy of S. aureus based on averting the immunogenicity of its dominant glycoantigen WTA. These results will help with the identification of invariant S. aureus vaccine antigens and may enable the development of TarP inhibitors as a new strategy for rendering MRSA susceptible to human host defences.

European external quality assessments for identification, molecular typing and characterization of Staphylococcus aureus.

Objectives: We present the results of two European external quality assessments (EQAs) conducted in 2014 and 2016 under the auspices of the Study Group on Staphylococci and Staphylococcal Infections of ESCMID. The objective was to assess the performance of participating centres in characterizing Staphylococcus aureus using their standard in-house phenotypic and genotypic protocols. Methods: A total of 11 well-characterized blindly coded S. aureus (n = 9), Staphylococcus argenteus (n = 1) and Staphylococcus capitis (n = 1) strains were distributed to participants for analysis. Species identification, MIC determination, antimicrobial susceptibility testing, antimicrobial resistance and toxin gene detection and molecular typing including spa typing, SCCmec typing and MLST were performed. Results: Thirteen laboratories from 12 European countries participated in one EQA or both EQAs. Despite considerable diversity in the methods employed, good concordance (90%-100%) with expected results was obtained. Discrepancies were observed for: (i) identification of the S. argenteus strain; (ii) phenotypic detection of low-level resistance to oxacillin in the mecC-positive strain; (iii) phenotypic detection of the inducible MLSB strain; and (iv) WGS-based detection of some resistance and toxin genes. Conclusions: Overall, good concordance (90%-100%) with expected results was observed. In some instances, the accurate detection of resistance and toxin genes from WGS data proved problematic, highlighting the need for validated and internationally agreed-on bioinformatics pipelines before such techniques are implemented routinely by microbiology laboratories. We strongly recommend all national reference laboratories and laboratories acting as referral centres to participate in such EQA initiatives.

Human NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) inflammasome activity is regulated by and potentially targetable through Bruton tyrosine kinase.

BACKGROUND: The Nod-like receptor NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) and Bruton tyrosine kinase (BTK) are protagonists in innate and adaptive immunity, respectively. NLRP3 senses exogenous and endogenous insults, leading to inflammasome activation, which occurs spontaneously in patients with Muckle-Wells syndrome; BTK mutations cause the genetic immunodeficiency X-linked agammaglobulinemia (XLA). However, to date, few proteins that regulate NLRP3 inflammasome activity in human primary immune cells have been identified, and clinically promising pharmacologic targeting strategies remain elusive. OBJECTIVE: We sought to identify novel regulators of the NLRP3 inflammasome in human cells with a view to exploring interference with inflammasome activity at the level of such regulators. METHODS: After proteome-wide phosphoproteomics, the identified novel regulator BTK was studied in human and murine cells by using pharmacologic and genetic BTK ablation. RESULTS: Here we show that BTK is a critical regulator of NLRP3 inflammasome activation: pharmacologic (using the US Food and Drug Administration-approved inhibitor ibrutinib) and genetic (in patients with XLA and Btk knockout mice) BTK ablation in primary immune cells led to reduced IL-1ß processing and secretion in response to nigericin and the Staphylococcus aureus toxin leukocidin AB (LukAB). BTK affected apoptosis-associated speck-like protein containing a CARD (ASC) speck formation and caspase-1 cleavage and interacted with NLRP3 and ASC. S aureus infection control in vivo and IL-1ß release from cells of patients with Muckle-Wells syndrome were impaired by ibrutinib. Notably, IL-1ß processing and release from immune cells isolated from patients with cancer receiving ibrutinib therapy were reduced. CONCLUSION: Our data suggest that XLA might result in part from genetic inflammasome deficiency and that NLRP3 inflammasome-linked inflammation could potentially be targeted pharmacologically through BTK.

Influence of Sae-regulated and Agr-regulated factors on the escape of Staphylococcus aureus from human macrophages.

Although Staphylococcus aureus is not a classical intracellular pathogen, it can survive within phagocytes and many other cell types. However, the pathogen is also able to escape from cells by mechanisms that are only partially understood. We analysed a series of isogenic S. aureus mutants of the USA300 derivative JE2 for their capacity to destroy human macrophages from within. Intracellular S. aureus JE2 caused severe cell damage in human macrophages and could efficiently escape from within the cells. To obtain this full escape phenotype including an intermittent residency in the cytoplasm, the combined action of the regulatory systems Sae and Agr is required. Mutants in Sae or mutants deficient in the Sae target genes lukAB and pvl remained in high numbers within the macrophages causing reduced cell damage. Mutants in the regulatory system Agr or in the Agr target gene psmα were largely similar to wild-type bacteria concerning cell damage and escape efficiency. However, these strains were rarely detectable in the cytoplasm, emphasizing the role of phenol-soluble modulins (PSMs) for phagosomal escape. Thus, Sae-regulated toxins largely determine damage and escape from within macrophages, whereas PSMs are mainly responsible for the escape from the phagosome into the cytoplasm. Damage of macrophages induced by intracellular bacteria was linked neither to activation of apoptosis-related caspase 3, 7 or 8 nor to NLRP3-dependent inflammasome activation.

Diversification of clonal complex 5 methicillin-resistant Staphylococcus aureus strains (Rhine-Hesse clone) within Germany.

Since 1995, a methicillin-resistant Staphylococcus aureus (MRSA) clone has spread in southern Germany. The strain was assigned to the Rhine-Hesse pulsed-field gel electrophoresis (PFGE) type by the staphylococcal reference center and was highly similar to epidemic clones known to belong to clonal complex 5 (CC5; USA100) based on multilocus sequence typing (MLST). Here we analyzed a defined collection of strains assigned to the Rhine-Hesse/USA100 PFGE type. Using sequence-based typing methods (MLST, spa), the isolates were divided into two distinct clusters, ST5 and its single-locus variant ST225. These two lineages are not distinguishable by PFGE or phage typing. Most of the ST5 isolates were derived from patients and volunteers from the Tübingen area in southwest Germany, whereas the ST225 isolates were mostly from other locations in Germany. The locally restricted ST5 isolates were shown to contain different SSCmec islands and exhibited different antibiotic resistance profiles. In contrast, the ST225 isolates form a highly homogenous group and are emerging all over Germany. The two lineages are clearly distinguishable by their phage content and spa type: ST5 strains from Tübingen are characterized by a Sa7int phage that carries the virulence gene sak, which codes for staphylokinase, and ST225 isolates are characterized by a Sa1int phage. In conclusion, based on sequence typing and phage content, CC5 strains can be subdivided into two distinct lineages with different epidemicities.

Import and spread of Panton-Valentine Leukocidin-positive Staphylococcus aureus through nasal carriage and skin infections in travelers returning from the tropics and subtropics.

BACKGROUND: Antibiotic-resistant Staphylococcus aureus is a globally emerging pathogen. Exchangeable virulence factors, such as Panton-Valentine leukocidin (PVL), have been proposed to drive this epidemic. We investigated whether skin infections and nasal colonization in travelers contribute to the global spread of such strains. METHODS: We conducted a case-control study of 38 returnees from the tropics and subtropics with S. aureus-positive skin and soft tissue infections (SSTIs) and 124 control patients with other travel-associated disorders. We collected information on travel characteristics, clinical outcomes of SSTIs, antibiotic sensitivity patterns, and genotypes of S. aureus strains isolated from skin lesions and the nares. RESULTS: S. aureus-positive SSTIs were associated with travel duration and purpose and were most common in returnees from Africa (odds ratio, 4.2; P = .005). PVL-positive (PVL(+)) S. aureus was frequent in the lesional and nasal isolates from travelers with SSTIs but could not be found in the nares of the control patients. The presence of PVL in S. aureus in travelers was associated with complicated disease, reduced antibiotic susceptibility, and secondary spread. The genotypes of PVL(+) S. aureus in returnees were reported to be endemic to the visited destination but rarely observed in Europe. CONCLUSIONS: Geographic variation in the risk of SSTIs in travelers supports a globally heterogeneous distribution of virulent S. aureus. Complicated SSTIs in returnees from nontemperate climates are associated with PVL(+) S. aureus and promote the emergence and spread of virulent and antibiotic-resistant strains. We propose a network for the surveillance of imported S. aureus (www.staphtrav.eu).

Characterization of an Enterococcus faecium small-colony variant isolated from blood culture.

Small-colony variants (SCVs) of bacteria are slow-growing subpopulations which can cause latent or recurrent infections due to better intracellular survival compared to their wild-type counterparts. Atypical colony morphology and altered biochemical profile may lead to failure in identification of SCV strains. We here report for the first time the isolation of an Enterococcus faecium SCV phenotype. The case of a 65-year-old woman with acute myeloid leukaemia who developed symptoms of sepsis during induction chemotherapy is presented. E. faecium with normal and SCV phenotype was isolated from blood cultures. At the same time urine culture was positive with E. faecium suggesting that bacteraemia originated from the urinary tract. The SCV phenotype was characterized by atypical growth behaviour. Electron microscopic analyses revealed perturbation of the separation of daughter cells and the accumulation of cell wall material. Accordingly, the SCV variant showed a dysfunction or lack of spontaneous autolysis whereas the normal phenotype did not. In contrast to conventional identification systems based on biochemical characteristics, the E. faecium SCV was precisely identified by MALDI-TOF MS analysis implemented in our laboratory. Hence, the increasing use of MALDI-TOF MS analysis for the identification of bacteria might be an appropriate tool for the detection of SCV variants, the diagnosis of which is of importance for the clinical outcome and the antibiotic treatment.

Diversity of prophages in dominant Staphylococcus aureus clonal lineages.

Temperate bacteriophages play an important role in the pathogenicity of Staphylococcus aureus, for instance, by mediating the horizontal gene transfer of virulence factors. Here we established a classification scheme for staphylococcal prophages of the major Siphoviridae family based on integrase gene polymorphism. Seventy-one published genome sequences of staphylococcal phages were clustered into distinct integrase groups which were related to the chromosomal integration site and to the encoded virulence gene content. Analysis of three marker modules (lysogeny, tail, and lysis) for phage functional units revealed that these phages exhibit different degrees of genome mosaicism. The prevalence of prophages in a representative S. aureus strain collection consisting of 386 isolates of diverse origin was determined. By linking the phage content to dominant S. aureus clonal complexes we could show that the distribution of bacteriophages varied remarkably between lineages, indicating restriction-based barriers. A comparison of colonizing and invasive S. aureus strain populations revealed that hlb-converting phages were significantly more frequent in colonizing strains.

Acquisition of antibiotic-resistant Enterococcus faecium strains during long-term hospitalization and fast adaptation of enterococcal flora to antibiotic treatment: a case report.

Recently, it has been suspected that long durations of hospitalization might be a possible risk factor to get colonized by multiple VRE strains. Here we present the case of a patient who underwent stem cell transplantation and subsequently stayed at the hospital for about 4 months until death. At least four different Enterococcus faecium strains were identified from routinely taken microbiological specimens as demonstrated by pulsed-field gel-electrophoresis. Additionally, these strains showed variable susceptibility to quinupristine/dalfopristine, vancomycin, and/or linezolid depending on different antibiotic administrations. These findings indicate that patients might be colonized with multiple Enterococcus faecium strains and that the enterococcal flora quickly adapts due to antibiotic exposure.

Linezolid-resistant E. faecium isolated from an open joint fracture: a report of the first isolate from Italy?

This report describes the isolation of a linezolid-resistant Enterococcus faecium and a carbapenem-resistant Acinetobacter baumannii from an infected open patella fracture after a car accident in southern Italy.

Relationship between suspected reasons of intraocular inflammation and the results of diagnostic vitrectomy: an observational study.

BACKGROUND: Intraocular inflammation of the posterior segment may be associated with neoplastic, infectious, and inflammatory diseases. Biopsy of vitreous and additional ocular tissue might be required for a definitive diagnosis. We therefore aimed to investigate the safety and usefulness of diagnostic vitrectomy in intraocular inflammation. METHOD: We performed an observational retrospective study of patients who underwent vitreous biopsy due to atypical intraocular inflammation, experienced failed resolution to empirical therapy, or for whom there was suspicion of an underlying neoplastic process. RESULTS: Seventy consecutive patients were included due to idiopathic uveitis of the posterior segment unresponsive to systemic corticosteroids (n = 33, 47%), suspected vitreoretinal or choroidal lymphoma (n = 18, 26%), viral retinitis (n = 17, 24%), and suspected endogenous endophthalmitis (n = 2, 3%). Suspected viral retinitis was most often confirmed (13/17, 76%), followed by lymphoma (6/18, 33%). Remarkably, a proportion of suspected idiopathic uveitis was infectious (7/33, 21%). The most prevalent adverse event following diagnostic vitrectomy was cataract surgery in phakic patients (n = 16/41, 39% of phakic patients). Retinal detachments were observed chiefly in infectious or neoplastic disease (n = 5, 7%) after a median time of 105 days. CONCLUSION: Diagnostic vitrectomy was helpful in substantiating the clinical suspected diagnosis of posterior segment inflammation. Potential secondary adverse events should be kept in mind.

Detection of pneumonia associated pathogens using a prototype multiplexed pneumonia test in hospitalized patients with severe pneumonia.

UNLABELLED: Severe pneumonia remains an important cause of morbidity and mortality. Polymerase chain reaction (PCR) has been shown to be more sensitive than current standard microbiological methods--particularly in patients with prior antibiotic treatment--and therefore, may improve the accuracy of microbiological diagnosis for hospitalized patients with pneumonia. Conventional detection techniques and multiplex PCR for 14 typical bacterial pneumonia-associated pathogens were performed on respiratory samples collected from adult hospitalized patients enrolled in a prospective multi-center study. Patients were enrolled from March until September 2012. A total of 739 fresh, native samples were eligible for analysis, of which 75 were sputa, 421 aspirates, and 234 bronchial lavages. 276 pathogens were detected by microbiology for which a valid PCR result was generated (positive or negative detection result by Curetis prototype system). Among these, 120 were identified by the prototype assay, 50 pathogens were not detected. Overall performance of the prototype for pathogen identification was 70.6% sensitivity (95% confidence interval (CI) lower bound: 63.3%, upper bound: 76.9%) and 95.2% specificity (95% CI lower bound: 94.6%, upper bound: 95.7%). Based on the study results, device cut-off settings were adjusted for future series production. The overall performance with the settings of the CE series production devices was 78.7% sensitivity (95% CI lower bound: 72.1%) and 96.6% specificity (95% CI lower bound: 96.1%). Time to result was 5.2 hours (median) for the prototype test and 43.5 h for standard-of-care. The Pneumonia Application provides a rapid and moderately sensitive assay for the detection of pneumonia-causing pathogens with minimal hands-on time. TRIAL REGISTRATION: Deutsches Register Klinischer Studien (DRKS) DRKS00005684.

Vancomycin-resistant enterococci outbreak, Germany, and calculation of outbreak start.

On the basis of a large outbreak of vancomycin-resistant Enterococcus faecium in a German university hospital, we estimated costs ( approximately 1 million Euros) that could have been avoided by early detection of the imminent outbreak. For this purpose, we demonstrate an easy-to-use statistical method.

Monoclonal outbreak of catheter-related bacteraemia by Ralstonia mannitolilytica on two haemato-oncology wards.

BACKGROUND: Ralstonia mannitolilytica is a non-fermentative, gram-negative bacterium isolated infrequently from clinical samples. However, within a period of 11 weeks five inpatients of the tertiary care hospital of the University of Tübingen developed clinical signs of infection and R. mannitolilytica was cultivated from blood samples of all patients suggesting an outbreak. METHODS: Blood cultures and one catheter tip were analysed by standard microbiological procedures. Genetic relatedness of the isolates was investigated by pulsed-field gel electrophoresis. To ascertain the possible source of the outbreak, environmental sampling and challenge-recovery experiments to test filters used for multi-dose solution bottles were performed. RESULTS: In the present study a monoclonal outbreak with R. mannitolilytica causing catheter-related infection of five haematological patients is reported. Underlying severe diseases with consecutive immunosuppression, permanent indwelling intravenous devices, multiple intravenous applications, and chemotherapy were possible risk factors promoting the infection. Challenge-recovery experiments revealed that R. mannitolilytica to a high extent even passed through Mini-spike Plus filters of pore size 0.2 microm. CONCLUSION: Although the source of the outbreak could not be identified, it is possible that solutions given intravenously were contaminated. Since R. mannitolilytica had never been isolated in our laboratory before and environmental testings performed were negative, it cannot be excluded that commercial products like drugs, saline solutions or infusion systems (filters) were contaminated.

Bartonella quintana variably expressed outer membrane proteins mediate vascular endothelial growth factor secretion but not host cell adherence.

Bartonella quintana causes trench fever, endocarditis, and the vasculoproliferative disorders bacillary angiomatosis and peliosis hepatis in humans. Little is known about the interaction of this pathogen with host cells. We attempted to elucidate the interaction of B. quintana with human macrophages (THP-1) and epithelial cells (HeLa 229). Remarkably, only B. quintana strain JK-31 induced secretion of vascular endothelial growth factor (VEGF) from THP-1 and HeLa 229 cells upon infection similar to the secretion induced by B. henselae Marseille, whereas other strains (B. quintana 2-D70, B. quintana Toulouse, and B. quintana Munich) did not induce such secretion. Immunofluorescence testing and electron microscopy revealed that the B. quintana strains unable to induce VEGF secretion did not express the variable outer membrane proteins (Vomps) on their surfaces. Surprisingly, the increase in VEGF secretion mediated by B. quintana JK-31 was not paralleled by elevated host cell adherence rates compared with the rates for Vomp-negative B. quintana strains. Our results suggest that the Vomps play a leading role in the angiogenic reprogramming of host cells by B. quintana but not in the adherence to host cells.