Molecular surveillance of carbapenem-resistant Enterobacterales in two nearby tertiary hospitals to identify regional spread of high-risk clones in Germany, 2019-2020.

BACKGROUND: Understanding the transmission dynamics of carbapenem-resistant Enterobacterales (CRE) is critical to addressing the escalating global threat of antimicrobial resistance (AMR). Although hospital transmission of CRE has been extensively studied, information on community transmission is lacking. AIM: To identify genomic clusters of CRE from two nearby institutions that may be indicative of community or inter-facility transmission. METHODS: CRE isolates between January 1st, 2019 and December 31st, 2020 from two tertiary hospitals, detected in the respective routine microbiology laboratories, were collected and characterized by short-read whole-genome sequencing. FINDINGS: A total of 272 CRE were collected, with Enterobacter cloacae complex (71/192, 37%) predominant in Heidelberg and Escherichia coli (19/80, 24%) in Mannheim. The most common carbapenem resistance gene, blaOXA-48, was detected in 38% of CRE from both centres. Several putative transmission clusters were found, including six clusters of E. cloacae complex, five clusters of Klebsiella pneumoniae, four clusters of Citrobacter freundii, and two clusters each of Escherichia coli and K. aerogenes. No clusters involved isolates from both study centres, except for an ST22 C. freundii cluster. Globally circulating clones were identified between the two centres for ST131 E. coli, ST66 E. hormaechei, and ST22 C. freundii. CONCLUSION: This study found no widespread transmission clusters among isolates from both centres, suggesting a hospital-specific clonal structure. This suggests that CRE clusters involving both institutions may indicate emerging or circulating clones in the community, highlighting the need for intersectoral surveillance and data sharing.

Cross-contamination of carbapenem-resistant Gram-negative bacteria between patients and the hospital environment in the first year of a newly built surgical ward.

BACKGROUND: Transmission and outbreaks of carbapenem-resistant Gram-negative bacteria (CRGN) in hospitals are often associated with contamination of the wastewater environment. We performed a prospective observational study to investigate the colonization of the hospital wastewater environment during the first year of occupancy of the surgical intermediate and intensive care units of a newly constructed building at the University Hospital of Heidelberg, Germany. METHODS: We performed monthly screening of the wastewater system (toilets and sinks) for 12 months, starting 1 month before opening (1st October 2020 to 30th October 2021). Admission and weekly rectal screening of patients for CRGN were also performed in parallel. Bacterial isolates were characterized by whole-genome sequencing. RESULTS: Twenty-seven of 1978 (1.4%) admitted patients were colonized/infected with CRGN. A total of 29 CRGN isolates from 24 patients and 52 isolates were available for sequencing. Within the first month of occupancy, we identified seven patients colonized/infected with CRGN, while none were found in the environmental reservoirs. The first detection of CRGN isolates in the sewage system started five months after the first occupancy. Two previously non-colonized patients were colonized/infected with Pseudomonas aeruginosa strains colonizing the sewage system. The significant identity of plasmids carrying the carbapenemase gene suggests that long-term colonization of the sewage system facilitates the emergence of new carbapenem-resistant clones. CONCLUSION: Cross-contamination between patients and the hospital environment is bidirectional. Our study demonstrated that contamination of the hospital wastewater environment may lead to persistent colonization and may serve as a reservoir for nosocomial acquisition of CRGN.

IL-1ß knockout increases the intestinal abundancy of Akkermansia muciniphila.

The proinflammatory cytokine interleukin-1ß (IL-1ß) is known to be upregulated in patients suffering from metabolic syndrome. IL-1ß contributes to insulin resistance in obesity and type 2 diabetes, yet its influence on the intestinal microbiome is incompletely understood. The data presented here demonstrate that mice genetically deficient in IL-1ß show a specific alteration of intestinal colonisation of a small group of bacteria. Especially Akkermansia muciniphila, a bacterium reported to be inversely associated with obesity, diabetes, cardiometabolic diseases and low-grade inflammation, showed increased colonisation in IL-1ß knockout mice. In comparative microarray analysis from mucus scrapings of the colon mucosa of IL-1ß knockout and wildtype mice, angiogenin 4 mRNA was strongly reduced in IL-1ß knockout animals. Since the presence of angiogenin 4 in the culture medium showed a significant growth inhibition on A. muciniphila which was not detectable for other bacteria tested, IL-1ß induced expression of angiogenin 4 is a strong candidate to be responsible for the IL-1ß induced suppression of A. muciniphila colonisation. Thus, the data presented here indicate that IL-1ß might be the lacking link between inflammation and suppression of A. muciniphila abundance as observed in a variety of chronic inflammatory disorders.

Impact of discontinuing contact precautions and enforcement of basic hygiene measures on nosocomial vancomycin-resistant Enterococcus faecium transmission.

OBJECTIVES: Vancomycin-resistant Enterococcus faecium (VREfm) has emerged as a pathogen of major concern for public health. Although definitive evidence is lacking, contact precautions have been a crucial element in infection prevention and control (IPC) strategies designed to limit nosocomial VRE transmission. This study investigated the effect of discontinuing contact precautions while enforcing basic hygiene measures, including hand hygiene, environmental cleaning and antiseptic body washing, for patients with VRE in intensive care units (ICUs) on the prevention of nosocomial VRE transmission causing bacteraemia. METHODS: Contact precautions were discontinued in January 2018. In total, 96 VREfm isolates from 61 patients with VREfm bacteraemia and/or colonization hospitalized in eight ICUs in a tertiary care hospital in 2016 and 2019 in were characterized by whole-genome sequencing. VRE transmission was investigated using patient movement data and admission screening for reliable identification of nosocomial acquisition. RESULTS: Discontinuation of contact precautions did not increase VREfm transmission events (eight in 2016 vs one in 2019). While the rate of endogenous VREfm was similar in both years (38% vs 31%), the number of non-colonized patients prior to VREfm bacteraemia was 16 (16/29, 55%) in 2019, which was significantly higher than in 2016 (8/32, 25%). The mean incidence density for VREfm bacteraemia was similar for both years (0.26 vs 0.31 per 1000 patient-days in 2016 and 2019, respectively). CONCLUSION: Discontinuation of contact precautions while enforcing basic hygiene measures did not lead to an increase in nosocomial bloodstream infection rates due to VREfm transmission in a hyperendemic ICU setting.

Whole-genome sequencing disproves two suspected transmission events of blaNDM between Pseudomonas aeruginosa and Enterobacterales in hospitalized patients.

New Delhi metallo-ß-lactamase (blaNDM) acquisition by Gram-negative bacteria is a primary concern due to its broad-host-range distribution. This study investigated two potential in-vivo horizontal gene transfers (HGTs) of blaNDM between Enterobacterales and Pseudomonas aeruginosa, initially indicated by polymerase chain reaction. Whole-genome sequencing showed independent parallel acquisition of two different blaNDM variants (NDM-1 and NDM-5) in P. aeruginosa and Enterobacterales, respectively. The data show that short-read sequencing provides the necessary resolution to confirm or dispute HGT by the comparison of genetic elements surrounding the gene of interest, and thus provide a timely response to potential outbreaks.

Alteration of antibiotic regimen as an additional control measure in suspected multi-drug-resistant Enterobacter cloacae outbreak in a neonatal intensive care unit.

BACKGROUND: Increased occurrence of a particular species of Gram-negative bacteria (GNB), especially when multi-drug-resistant (MDR), in routine screening surveillance in neonatal intensive care units (NICUs) can be evoked by selection pressure. AIM: To evaluate adaptation of the empiric antibiotic regimen for its usefulness as a control measure in suspected outbreaks in the NICU. METHODS: In a retrospective outbreak analysis, cases between 1st December 2017 and 31st March 2018 were identified through microbiology and hygiene surveillance records. Furthermore, risk factors for MDR-GNB colonization were collected. Whole-genome sequencing (WGS) was performed on all isolates. Control measure documentations and interviews were employed to define interventions. As well as infection control measures, administration of third-generation cephalosporins was avoided and replaced whenever clinically acceptable as part of the intervention bundle. FINDINGS: In total, nine patients were found to have rectal colonization with third-generation cephalosporin-resistant Enterobacter cloacae in routine screening surveillance in the pre-intervention period. After implementation of an infection control bundle, the incidence declined rapidly. WGS analysis revealed that two MDR E. cloacae were transmitted, and the majority were new cases. The incidence density of MDR-GNB colonization was 7.94/1000 patient-days (PD) before the intervention and 1.68/1000 PD during the altered antibiotic regimen. No infections with MDR-GNB occurred during the study. CONCLUSIONS: Altering the antibiotic regimen with regard to selection pressure may be considered as part of an intervention bundle to rapidly control the emergence of MDR-GNB in suspected outbreak situations in the NICU.

Comparative genomic analysis reveals a high prevalence of inter-species in vivo transfer of carbapenem-resistance plasmids in patients with haematological malignancies.

OBJECTIVES: Conjugative gene transfer has been considered as one of the driving factors in the transmission and dissemination of multidrug resistance in bacteria. The abundance of antimicrobial resistance genes and bacteria in the gut microbiome may provide the ideal platform for plasmid exchange. Systematic data on in vivo horizontal gene transfer (HGT) and its frequency are scarce. MATERIALS AND METHODS: One hundred and ninety-six carbapenem-resistant gram-negative bacilli (CRGNBs) from 179 patients (158 inpatients and 21 outpatients) between January 2016 and April 2017 were analysed retrospectively. Alignment of plasmid content for 32 isolates from 16 patients with multiple CRGNB species was performed from whole-genome sequencing (WGS) data. RESULTS: Sixteen of the 179 patients (8.9%) were colonized and/or infected with more than one CRGNB species; 11/179 (6.1%) were colonized by multiple carbapenem-resistant Enterobacteriaceae (CREs) and 5/179 (2.8%) by carbapenem-resistant non-fermenters (CRNFs) and CREs. WGS suggested interspecies transfer as the predominant mechanism rather than independent acquisition in 8/10 patients (80%, one non-recoverable isolate) with multiple CREs but not in CRNF-CRE combinations; 30/158 inpatients (20%) had underlying haematological malignancies, and they are more likely to exhibit multiple CRGNB strains (OR 3.0, 95%CI 0.98-8.89, p 0.05) and CRE strains (OR 3.9, 95%CI 1.02-14.58, p 0.04) during hospital stay compared to other patient groups. CONCLUSION: Our data give insight into the occurrence of natural in vivo HGT in a clinical setting. Better understanding of HGT will help optimize containment measures and may guide antibiotic stewardship programmes.

Import of community-associated, methicillin-resistant Staphylococcus aureus to Europe through skin and soft-tissue infection in intercontinental travellers, 2011-2016.

OBJECTIVES: Recently, following import by travel and migration, epidemic community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has caused nosocomial outbreaks in Europe, sometimes with a fatal outcome. We describe clinico-epidemiological characteristics of CA-MRSA detected by the European Network for the Surveillance of imported S. aureus (www.staphtrav.eu) from May 2011 to November 2016. METHODS: Sentinel surveillance at 13 travel clinics enrolling patients with travel-associated skin and soft-tissue infection (SSTI) and analysing lesion and nose swabs at one central laboratory. RESULTS: A total of 564 independent case-patients with SSTI were enrolled and had 374 (67%) S. aureus-positive lesions, of which 14% (51/374) were MRSA. The majority of CA-MRSA isolates from SSTI were Panton-Valentine leucocidin (PVL) -positive (43/51, 84%). The risk of methicillin-resistance in imported S. aureus varied by travel region (p <0.001) and was highest in Latin America (16/57, 28%, 95% CI 17.0-41.5) and lowest in Sub-Saharan Africa (4/121, 3%, 95% CI 0.9-8.3). Major epidemic clones (USA300 / USA300 Latin-American Variant, Bengal Bay, South Pacific) accounted for more than one-third (19/51, 37%) of CA-MRSA imports. CA-MRSA SSTI in returnees was complicated (31/51 multiple lesions, 61%; 22/50 recurrences, 44%), led to health-care contact (22/51 surgical drainage, 43%; 7/50 hospitalization, 14%), was transmissible (13/47 reported similar SSTI in non-travelling contacts, 28%), and associated with S. aureus nasal colonization (28 of 51 CA-MRSA cases, 55%; 24 of 28 colonized with identical spa-type in nose and lesion, 85%). CONCLUSIONS: Travel-associated CA-MRSA SSTI is a transmissible condition that leads to medical consultations and colonization of the infected host.

Toll-like receptor 9 (TLR-9) promotor polymorphisms and gene expression are associated with persistent Staphylococcus aureus nasal carriage.

OBJECTIVES: Toll-like receptor (TLR) 9 could have importance in human Staphylococcus aureus immunity, but population-level evidence for this hypothesis is missing. METHODS: We phenotyped S. aureus nasal carriage of 603 volunteers using four consecutive swabs, genotyped TLR9 promotor variants in 106 persistent carriers and 219 noncarriers, measured TLR9-mRNA expression in whole blood after stimulation with viable S. aureus and studied mutual associations of carriage, transcriptional activity and single nucleotide polymorphisms while accounting for sex and hormone contraceptive use (HCU). RESULTS: The -1486 (rs187084) and -1237 (rs5743836) CT haplotype was more common in noncarriers (185/438, 42%) than in carriers (63/212, 30%), with the TT haplotype showing a reverse association (noncarriers, 180/438, 41%; carriers 117/212, 55%) (χ2 p 0.001). Mean TLR9 mRNA expression in whole blood was higher in noncarriers (ratiocarriers/noncarriers 0.63; 95% confidence interval, 0.43-0.92; p 0.017). A duplication of TLR9 transcriptional activity lowered the odds of persistent S. aureus carriage by 37% in the overall group (odds ratio = 0.63; 95% confidence interval, 0.42-0.94; p 0.022) and by 54% in women (odds ratio = 0.46; 95% confidence interval, 0.23-0.90; p 0.023). Promotor haplotype and HCU had a combined effect on TLR9 transcription (interaction model): women in the TT (risk) haplotype/HCU- stratum (baseline) had lower mRNA levels than women in the CT (protective) haplotype/HCU- (ratio 1.92; p 0.055), the CT haplotype/HCU+ (ratio 2.02; p 0.032) and the TT haplotype/HCU+ (ratio 2.59; p < 0.004) strata. No such associations were observed in men. CONCLUSIONS: We provide evidence that TLR9 affects human S. aureus immunity and present potential explanations for differences according to sex in S. aureus colonization and infection.

Spectral narrowing of x-ray pulses for precision spectroscopy with nuclear resonances.

Spectroscopy of nuclear resonances offers a wide range of applications due to the remarkable energy resolution afforded by their narrow linewidths. However, progress toward higher resolution is inhibited at modern x-ray sources because they deliver only a tiny fraction of the photons on resonance, with the remainder contributing to an off-resonant background. We devised an experimental setup that uses the fast mechanical motion of a resonant target to manipulate the spectrum of a given x-ray pulse and to redistribute off-resonant spectral intensity onto the resonance. As a consequence, the resonant pulse brilliance is increased while the off-resonant background is reduced. Because our method is compatible with existing and upcoming pulsed x-ray sources, we anticipate that this approach will find applications that require ultranarrow x-ray resonances.

Interferometric phase detection at x-ray energies via Fano resonance control.

Modern x-ray light sources promise access to structure and dynamics of matter in largely unexplored spectral regions. However, the desired information is encoded in the light intensity and phase, whereas detectors register only the intensity. This phase problem is ubiquitous in crystallography and imaging and impedes the exploration of quantum effects at x-ray energies. Here, we demonstrate phase-sensitive measurements characterizing the quantum state of a nuclear two-level system at hard x-ray energies. The nuclei are initially prepared in a superposition state. Subsequently, the relative phase of this superposition is interferometrically reconstructed from the emitted x rays. Our results form a first step towards x-ray quantum state tomography and provide new avenues for structure determination and precision metrology via x-ray Fano interference.

Risk factors and containment of respiratory syncytial virus outbreak in a hematology and transplant unit.

Respiratory syncytial virus (RSV) usually causes self-limiting upper respiratory tract infections, but can be associated with severe lower respiratory tract infection disease (LRTID) in infants and in patients with hematologic malignancies. We have analyzed the risk factors and the measures for containment within an outbreak of nosocomial RSV infections in a hematology and SCT unit. A total of 56 patients were affected (53 RSV-A and 3 RSV-B) including 32 transplant patients (16 allogeneic and 16 autologous). Forty (71%) of the 56 patients suffered from LRTID and 14 (35%) of the patients with LRTID subsequently died. However, because of concomitant infections with fungal and bacterial pathogens, the impact of RSV on the fatal outcome was difficult to assess. Multivariate analysis showed that low levels of IgG were significantly associated with fatal outcome (P=0.007), treatment with oral ribavirin represented a protective factor (P=0.02). An extremely protracted viral shedding was observed in this cohort of patients (median=30.5 days, range: 1-162 days), especially pronounced in patients after allogeneic transplantation (P=0.002). Implementation of rigorous isolation and barrier measures, although challenged by long-term viral carriers, was effective in containment of the outbreak.

Effectiveness of low-intensity endurance training.

Recent studies point to the preventive efficacy of low-intensity endurance training in terms of cardiovascular risk factor modification and mortality reduction. In addition, it is frequently recommended as a means of stimulating fat metabolism. It was the intention of this study to clarify if endurance training effectiveness remains unimpaired when exercise intensity is reduced by a certain amount from "moderate" to "low", but total energy expenditure held constant. For this purpose, 39 healthy untrained subjects (44 +/- 7 yrs, 82 +/- 19 kg; 173 +/- 9 cm) were stratified for endurance capacity and sex and randomly assigned to 3 groups: "moderate intensity" (MOD, n = 13, 5 sessions per week, 30 min each, intensity 90 % of the anaerobic threshold [baseline lactate + 1.5 mmol/l]), "low intensity" (LOW, n = 13, 5 sessions per week, intensity 15 bpm below MOD, duration proportionally longer to arrive at the same total energy output as MOD), and control (CO, n = 13, no training). Training was conducted over 12 weeks and each session monitored by means of portable heart rate (HR) recorders. Identical treadmill protocols prior to and after the training program served for exercise prescription and documentation of endurance effects. VO (2max) improved similarly in both training groups (MOD + 1.5 ml x min (-1) x kg (-1); LOW + 1.7 ml x min (-1) x kg (-1); p = 0.97 between groups). Compared with CO (- 1.0 ml x min (-1) x kg (-1)) this effect was significant for LOW (p < 0.01) whereas there was only a tendency for MOD (p = 0.07). However, objective criteria (HR (max), maximal blood lactate) indicated that a different degree of effort was responsible for this finding. In comparison with CO (mean decrease of 3 bpm), average HR during incremental exercise decreased significantly by 9 bpm (MOD, p < 0.05 vs. CO) and 6 bpm (LOW, p = 0.26), respectively. However, there was no significant difference between MOD and LOW (p = 0.60), but for changes in oxygen uptake at the anaerobic threshold (VO (2AT)) it was observed that MOD was significantly more effective than CO (p = 0.048) and LOW (p = 0.04). It is concluded that within a middle-aged population of healthy untrained subjects, endurance training effectiveness might be slightly impaired when the training heart rate is chosen 15 bpm lower as compared to moderate intensity, but the total energy output held equal.

Evaluation of a single-locus real-time polymerase chain reaction as a screening test for specific detection of methicillin-resistant Staphylococcus aureus in ICU patients.

The aim of the present study was to determine the diagnostic value of a single-locus real-time polymerase chain reaction (PCR) recently proposed for rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) from clinical samples (IDI-MRSA; Infectio Diagnostic, Sainte-Foy, Québec, Canada). This test, which was developed on the basis of studies of the sequence analysis of the mecA gene carried by staphylococcal cassette chromosome mec (SCCmec), was used to screen nasal swabs of 320 intensive care unit (ICU) patients at admission. The results were compared with those of conventional culture of swabs from several body sites. When compared with culture of swabs from the nose, throat, and wounds, the diagnostic values of the real-time PCR test from nasal swabs were as follows: 92.3% sensitivity, 98.6% specificity, 75.0% positive predictive value, and 99.6% negative predictive value. Fifteen (4.7%) samples could not be evaluated because the PCR reaction was inhibited, even after the samples were frozen and thawed for retesting. Culture of nasal swabs showed that 78 of the patients were colonized with methicillin-susceptible S. aureus. Unexpectedly, 4 (5.1%) of these samples gave false-positive results in the IDI-MRSA. These isolates were all single clones, as shown by pulsed-field gel electrophoresis and spa typing. Reliable results were obtained with the IDI-MRSA assay, even in a patient population with a low prevalence (approximately 4%) of MRSA and even when compared with swabs of different body sites. Nevertheless, further work is needed to reduce the inhibition rate of the PCR and to explain why false-positive results were obtained with methicillin-susceptible S. aureus.

Immunostimulatory CpG oligonucleotides reduce tumor burden after intravesical administration in an orthotopic murine bladder cancer model.

Bacillus Calmette-Guérin is established in the prophylaxis of recurrent intermediate and high-risk superficial bladder cancer and induces an unspecific, Th1-biased local immune response. Small CpG oligonucleotides (CpG ODN) containing a central unmethylated CpG motif are able to mimic the immunostimulatory activity of bacterial DNA. The purpose of the present study was to evaluate the antineoplastic properties of intravesically administered CpG ODN in an orthotopic murine bladder cancer model. MB49 tumor cell suspension was instilled transurethrally in female C57/BL6 mice on day 0. Mice were divided in three groups of 12 animals. Four mice in each group received either stimulative CpG ODN, non-stimulative GpC ODN or PBS intravesically: group I on day 3, group II on day 5, group III on day 7. After sacrifice 7 days after treatment, bladders were removed and histological examinations were performed. Single instillation of CpG ODN revealed antineoplastic effects in every group demonstrated by significantly lower bladder weight compared with non-stimulative GpC ODN- and PBS-treated mice. Histological examination showed extensive infiltration of macrophages and lymphocytes in CpG ODN-treated mice, whereas PBS- and GpC ODN-treated mice showed solid tumor growth with only few leucocytes. Intravesically applied immunostimulative DNA demonstrated antitumoral activity in an orthotopic murine bladder cancer model. A single instillation seems to be sufficient to reduce tumor load.

Deficiency in immunoglobulin G2 antibodies against staphylococcal enterotoxin C1 defines a subgroup of patients with atopic dermatitis.

BACKGROUND AND AIM: Atopic dermatitis (AD) is a common chronic inflammatory skin disease often accompanied by cutaneous Staphylococcus aureus colonization and, in this regard, especially complicated by the presence of superantigen-producing strains. Because IgG antibodies comprise an important defence mechanism of the adaptive immune system against bacteria, it was investigated whether AD patients have an abnormal pattern or distribution of superantigen-specific IgG subclass antibodies in association with disease severity and activity. METHODS: Staphylococcal enterotoxin B (SEB) and staphylococcal enterotoxin C1 (SEC1) specific IgG antibody subclasses were assessed in n=89 adult AD patients with mild to severe disease activity as determined by the SCORAD score and in n=28 healthy age-matched controls. Results were correlated with the current status of bacterial skin colonization and severity score. RESULTS: Thirty-eight per cent of the AD patients showed a selective deficiency in IgG2 antibodies against SEC1 compared with only 14% in the control group. The absence of these antibodies was found in both currently colonized and non-colonized AD patients and was associated with a severe phenotype (SCORAD more than 40 points in two-thirds of the deficient patients). However, these patients had normal production levels of IgG2 antibodies against pneumococcal capsular polysaccharide (PCP) and SEB, but higher IgG1 and IgG4 titres against SEC1. Except for elevated total IgG1, total IgG subclass levels were normal in this AD subgroup. Yet, peripheral blood mononuclear cells (PBMCs) derived from these patients clearly produced IL-4 and IL-5 upon SEC1 re-stimulation whereas PBMCs from those providing SEC1-specific IgG2 antibodies failed in the production of these cytokines. CONCLUSION: A subgroup of AD patients suffers from a selective deficiency to produce anti-SEC1 IgG2 antibodies. This patient group is characterized by a severe AD phenotype.

Detection of Staphylococcus aureus enterotoxins A to D by real-time fluorescence PCR assay.

Staphylococcus aureus is one of the most significant pathogens causing nosocomial and community-acquired infections. Among the secreted staphylococcal virulence factors, there is a growing list of enterotoxins which can induce gastroenteric syndrome and toxic shock syndrome. Here, we developed a real-time fluorescence PCR assay (TaqMan PCR) for the detection of genes encoding staphylococcal enterotoxins A, B, C1, and D (SEA, SEB, SEC1, and SED) of S. aureus as well as the mecA gene encoding methicillin resistance and the femB gene as a specific genomic marker for S. aureus. SEA to SED were selected because they are the four classically described enterotoxins of S. aureus and because they were detected by latex agglutination. In order to evaluate the reliability of TaqMan PCR, we investigated 93 isolates of S. aureus derived from patients at our hospital over 5 months and compared the results with data obtained by a commercially available reversed passive latex agglutination assay (SET-RPLA) for these isolates. Thirteen enterotoxin genes were detected by TaqMan PCR; however, no proteins expressed by these genes were detected by SET-RPLA. As a result, more isolates of S. aureus (n = 44) were found positive by TaqMan PCR for one or more enterotoxin genes than by SET-RPLA for the respective proteins expressed by these genes (n = 40). We conclude that TaqMan PCR is more sensitive because it offers the possibility for determining enterotoxins on a genotypic basis. Additionally, the assay allows the parallel detection of genes for SEA to SED and methicillin resistance in S. aureus. Furthermore, real-time PCR is well suited for screening large numbers of samples at the same time, allowing rapid, reliable, efficient, and cost-saving routine laboratory diagnosis.

IL-4 instructs TH1 responses and resistance to Leishmania major in susceptible BALB/c mice.

Immunity to infection with intracellular pathogens is regulated by interleukin 12 (IL-12), which mediates protective T helper type 1 (TH1) responses, or IL-4, which induces TH2 cells and susceptibility. Paradoxically, we show here that when present during the initial activation of dendritic cells (DCs) by infectious agents, IL-4 instructed DCs to produce IL-12 and promote TH1 development. This TH1 response established resistance to Leishmania major in susceptible BALB/c mice. When present later, during the period of T cell priming, IL-4 induced TH2 differentiation and progressive leishmaniasis in resistant mice. Because immune responses developed via the consecutive activation of DCs and then T cells, the contrasting effects of IL-4 on DC development and T cell differentiation led to immune responses that had opposing functional phenotypes.

Suppressors of cytokine signaling (SOCS)-1 and SOCS-3 are induced by CpG-DNA and modulate cytokine responses in APCs.

During infection, the functional status of the innate immune system is tightly regulated. Although signals resulting in activation have been well characterized, counterregulative mechanisms are poorly understood. Suppressor of cytokine signaling (SOCS) proteins have been characterized as cytokine-inducible negative regulators of Janus kinase/STAT signaling in cells of hemopoietic origin. To analyze whether SOCS proteins could also be induced by pathogen-derived stimuli, we investigated the induction of SOCS-1 and SOCS-3 after triggering of macrophage cell lines, bone marrow-derived dendritic cells, and peritoneal macrophages with CpG-DNA. In this study, we show that CpG-DNA, but not GpC-DNA, induces expression of mRNA for SOCS-1 and SOCS-3 in vitro and in vivo. SOCS mRNA expression could be blocked by chloroquine and was independent of protein synthesis. Inhibitors of the mitogen-activated protein kinase pathway triggered by CpG-DNA were able to impede induction of SOCS mRNA. CpG-DNA triggered synthesis of SOCS proteins that could be detected by Western blotting. SOCS proteins were functional because they inhibited IFN-gamma as well as IL-6- and GM-CSF-induced phosphorylation of STAT proteins. Furthermore, IFN-gamma-induced up-regulation of MHC class II molecules was also prevented. The same effects could be achieved by overexpression of SOCS-1. Hence, the results indicate a substantial cross-talk between signal pathways within cells. They provide evidence for regulative mechanisms of Janus kinase/STAT signaling after triggering Toll-like receptor signal pathways.