Course of colonization by multidrug-resistant organisms after allogeneic hematopoietic cell transplantation.

Multidrug-resistant organisms (MDRO) have been developing as an emerging problem in allogeneic hematopoietic cell transplantation (HCT). Since no data are available on the course of MDRO colonization after HCT, we investigated in this retrospective, single-center study, persistence and clearance of MDRO after HCT. From June 2010 to December 2015, 121 consecutive HCT patients were included. Patients received a MDRO screening before conditioning as well as surveillance cultures after HCT. In MDRO-colonized patients, surveillance specimens were taken until MDRO were no longer detectable. Thirty-three patients (27%) were found to be colonized by at least one MDRO at any time point until day 100 post HCT. Day 100 (2-year) non-relapse mortality (NRM) and overall survival (OS) of MDRO-colonized (MDRO+) versus non-colonized (MDRO-) patients were essentially the same. NRM is 15% (21%) versus 15% (24%). Two-year OS is 60 versus 55% for MDRO+ versus MDRO- patients. Out of the 33 MDRO+ patients, 21 cleared the MDRO. Median time to non-detectability of MDRO was 6 months. In 12 patients, the MDRO persisted. There was a significant (p < 0.0001) survival difference between patients who cleared the MDRO versus those with MDRO persistence (2-year OS 80 vs 40%). Except for the length of antibiotic therapy as a potential risk factor for MDRO persistence after HCT, no other conventional factors could be identified. (a) colonization by MDRO per se had no negative impact on the outcome, (b) MDRO can be cleared by the majority of patients after allogeneic HCT, and (c) to increase the probability to clear MDRO, the use of antibiotics in MDRO+ patients should be reviewed critically.

Rapid detection of cefotaxime-resistant Escherichia coli by LC-MS.

Antibiotic resistance is an unsolved healthcare problem with increasing impact on patient management in the last years. In particular, multidrug resistance among Gram-negative bacterial strains has become the most pressing challenge. In order to deliver the most efficacious antimicrobial therapy with minimum delay, rapid diagnostic tests are required in order to detect multidrug resistant pathogens early during infection. In line with these efforts, we have developed a mass spectrometry-based assay for the rapid determination of ampicillin and cefotaxime resistance. The assay quantifies beta-lactamase activities towards ampicillin and cefotaxime within a turnaround time of 150 min, which is substantially faster than classical susceptibility testing.

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.

Rapid detection of ampicillin resistance in Escherichia coli by quantitative mass spectrometry.

Early targeted antimicrobial therapy helps decrease costs and prevents the spread of antimicrobial resistance, including in Escherichia coli, the most frequent Gram-negative bacterium that causes sepsis. Therefore, rapid susceptibility testing represents the major prerequisite for knowledge-based successful antimicrobial treatment. To accelerate testing for antibiotic susceptibility, we have developed a new mass spectrometry-based assay for antibiotic susceptibility testing (MAAST). For proof of principle, we present an ampicillin susceptibility test for E. coli with a turnaround time of 90 min upon growth detection.

[Increasing therapeutic challenges through multi-resistant bacteria in the hospital]. / Zunehmende therapeutische Herausforderungen durch multiresistente Keime in der Klinik.

BACKGROUND: Multi-resistant bacteria endanger with increasing frequency the successful outcome of antibiotic therapy, which represents the presumably most successful discovery of medicine. Due to a worldwide misuse of antibiotics and combined with a lacking awareness of hospital hygiene, multi-resistant bacteria spread with a worrisome pace within the clinic. These bacteria also spread globally via modern transportation systems. Today bacteria are present in hospitals, which are sensitive to a rather limited number of antibiotics or even -worse are pan-resistant. The growing threat of multi-resistant bacteria is further increased by the fact that the pipeline of the pharmaceutical industry for new antibiotics is more or less empty. Furthermore, economical pressure will increase the workload for hospital employees and by that support the spread of -multi-resistant bacteria. In addition, patients will be more susceptible to infections since on the one hand they are on average of older age and on the other hand treated with more aggressive surgical or immuno-suppressive regimens. CONCLUSION: This threatening but realistic future scenario can only be avoided by a combination of measures: absolute justified application of antibiotics, a consequent use of standard hygiene, -isolation of contaminated or infected patients and sufficient employees and their education in the prevention of infections.

Chemokine response induced by Chlamydia trachomatis in prostate derived CD45+ and CD45- cells.

The role of innate cells and their receptors within the male genital tract remains poorly understood. Much less is known about the relative contribution of different genital tract cells such as epithelial/stromal cells and resident leucocytes. In this study, we examined innate immune responses to Chlamydia trachomatis by prostate epithelial/stromal cells and prostate resident leucocytes. Murine prostate primary cultures were performed and leucocyte and epithelial/stromal cells were sorted based on surface protein expression of CD45 by magnetism-activated cell sorting or fluorescence-activated cell sorting. Prostate derived CD45- and CD45+ cells were infected with C. trachomatis and chemokine secretion assayed by ELISA. Similar experiments were performed using prostate CD45+ and CD45- cells from myeloid differentiation factor 88 (Myd88(-/-)) mice or toll-like receptor (Tlr2(-/-)) and Tlr4(mutant) double-deficient mice. Moreover, a TLR-signalling pathway array was used to screen changes in different genes involved in TLR-signalling pathways by real-time PCR. Prostate derived CD45- and CD45+ cells responded to chlamydial infection with the production of different chemokines. Both populations expressed genes involved in TLR signalling and required to respond to pathogen-associated molecular patterns and to C. trachomatis infection. Both populations required the adaptor molecule MYD88 to elicit chemokine response against C. trachomatis. TLR2-TLR4 was essential for chemokine production by CD45+ prostate derived cells, but in their absence, CD45- cells still produced significant levels of chemokines. We demonstrate that C. trachomatis is differentially recognised by prostate derived CD45+ and CD45- cells and suggest that diverse strategies are taking place in the local microenvironment of the host in response to the infection.

[Antibiotic prophylaxis in primary and revision hip arthroplasty: what is the evidence?]. / Antibiotische Infektionsprophylaxe in der Primär- und Revisionsendoprothetik der Hüfte: Was ist erwiesen?

Advances in the perioperative and postoperative management of total joint replacement have led to a steady decrease in the infection rate, which in the case of total hip replacement presently lies between 0.25 and 1%. Unfortunately there is disparity in current practice nationally and internationally, regarding duration, time of application and choice of antibiotics. Currently there are only Level 1a recommendations for primary hip arthroplasty, whereas, due to the heterogeneity and complexity of most revision cases as well as a lack of randomized controlled trials, antibiotic prophylaxis for hip revision arthroplasty is mostly based on the surgeon's preference. In this article the current literature is reviewed and scientifically sound data and recommendations are summarized.

[Results of preoperative SARS-CoV-2 testing in the coronavirus pandemic]. / Ergebnisse der präoperativen SARS-CoV-2-Testung ("severe acute respiratory syndrome coronavirus 2") in der Coronaviruspandemie.

BACKGROUND: Surgery is challenging during the coronavirus disease 2019 (COVID-19) pandemic. This study aimed to investigate the preoperative severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing in elective and emergency surgery and to calculate the patient contacts during hospital stay. MATERIALS AND METHODS: All surgeries defined by the German procedural classification (starting with a 5) from 1 June until 29 November 2020 were retrospectively evaluated regarding the preoperative SARS-CoV­2 nasopharyngeal swab test. The results were then divided in emergency and elective surgeries. To show the personal contacts of the patients in a university hospital, we calculated the patient pathway within the department of urology and urosurgery for April 2020. Therefor we used the electronic patient records. RESULTS: Altogether 7745 surgical procedures in 5985 patients were performed, whereby 39 (0.5%) SARS-CoV­2 tests were positive. 2833 (37%) surgical procedures were emergency cases and 4912 (63%) were elective procedures. 25 (0.9%) of the emergency group and 14 (0.3%) of the elective surgeries had a positive SARS-CoV­2 test. The average number of contacts in the patient room was 12.83 (0-50) and 84.22 (0-249) at the ward level, not counting contacts with the clinic staff. CONCLUSIONS: Nearly 1% of the preoperative SARS-CoV­2 tests of either emergency or elective surgeries tested positive in the 6 months prior to November 2020. Although the risk of undetected SARS-CoV­2 infection appears to be low in terms of costs and personnel, preoperative screening is useful in high-risk areas to ensure further necessary surgeries, especially concerning cancer patients and to prevent virus spread in a hospital.

T cell-mediated lethal shock triggered in mice by the superantigen staphylococcal enterotoxin B: critical role of tumor necrosis factor.

Because mice are more resistant than humans to the pathogenic effects of bacterial toxins, we used D-Galactosamine- (D-Gal) sensitized mice as a model system to evaluate potential toxic shock symptoms triggered by the superantigen staphylococcal enterotoxin B (SEB). We show that similar to endotoxin (lipopolysaccharide) [LPS], the exotoxin SEB causes lethal shock within 8 h in D-Gal-sensitized mice, inducing 100% and about 50% lethality with 20 and 2 micrograms SEB, respectively. The lethal shock triggered by the superantigen SEB is mediated by T cells, a conclusion based on the observation that T cell repopulation of SCID mice conferred sensitivity to SEB. Since CSA also conferred protection, the role of T cell-derived lymphokines in mediating lethal shock was evaluated. Within 30-60 min after SEB injection, serum tumor necrosis factor (TNF) levels peaked, followed immediately by interleukin-2 (IL-2). Serum-borne lymphokines were detected well in advance of signs of T cell activation, as assessed by IL-2 receptor expression of SEB-reactive V beta 8+ T cells. Passive immunization with anti-TNF-alpha/beta-neutralizing monoclonal antibody also conferred protection, indicating that it is TNF which is critical for initiating toxic shock symptoms. Taken together, this study defines basic differences between endotoxin (LPS)- and exotoxin (SEB)-mediated lethal shock, in that the former is mediated by macrophages and the latter by T cells. Yet the pathogenesis distal to the lymphokine/cytokine-producing cells appears surprisingly similar in that TNF represents a key mediator in inducing shock.

Effectiveness of ozone against endodontopathogenic microorganisms in a root canal biofilm model.

AIM: To assess the antimicrobial efficacy of aqueous (1.25-20 microg mL(-1)) and gaseous ozone (1-53 g m(-3)) as an alternative antiseptic against endodontic pathogens in suspension and a biofilm model. METHODOLOGY: Enterococcus faecalis, Candida albicans, Peptostreptococcus micros and Pseudomonas aeruginosa were grown in planctonic culture or in mono-species biofilms in root canals for 3 weeks. Cultures were exposed to ozone, sodium hypochlorite (NaOCl; 5.25%, 2.25%), chlorhexidine digluconate (CHX; 2%), hydrogen peroxide (H(2)O(2); 3%) and phosphate buffered saline (control) for 1 min and the remaining colony forming units counted. Ozone gas was applied to the biofilms in two experimental settings, resembling canal areas either difficult (setting 1) or easy (setting 2) to reach. Time-course experiments up to 10 min were included. To compare the tested samples, data were analysed by one-way anova. RESULTS: Concentrations of gaseous ozone down to 1 g m(-3) almost and aqueous ozone down to 5 microg mL(-1) completely eliminated the suspended microorganisms as did NaOCl and CHX. Hydrogen peroxide and lower aqueous ozone concentrations were less effective. Aqueous and gaseous ozone were dose- and strain-dependently effective against the biofilm microorganisms. Total elimination was achieved by high-concentrated ozone gas (setting 2) and by NaOCl after 1 min or a lower gas concentration (4 g m(-3)) after at least 2.5 min. High-concentrated aqueous ozone (20 microg mL(-1)) and CHX almost completely eliminated the biofilm cells, whilst H(2)O(2) was less effective. CONCLUSION: High-concentrated gaseous and aqueous ozone was dose-, strain- and time-dependently effective against the tested microorganisms in suspension and the biofilm test model.

Management of facial necrotizing fasciitis.

Necrotizing fasciitis is a progressive, life-threatening, bacterial infection of the skin, the subcutaneous tissue and the underlying fascia, in most cases caused by ss-hemolytic group A streptococcus. Only early diagnosis and aggressive therapy including broad spectrum antibiotics and surgical intervention can avoid systemic toxicity with a high mortality rate. This uncommon disease generally occurs in the lower extremities and trunk, and only rarely affects the head and neck region. When located in the face necrotizing fasciitis is associated with severe cosmetic and functional restrictions due to the invasive infection and often to the necessary surgical treatment. Generally surgical intervention cannot be performed in the face as aggressively as in the extremities and trunk, since a lot of vital structures are found in a relatively small area. In the following article, we present the successful diagnostic and therapeutic management of an isolated facial necrotizing fasciitis as a consequence of a nasal bone fracture with a minor dermal cut.

Previous cytomegalovirus infection and restenosis after coronary stent placement.

BACKGROUND: Reactivated cytomegalovirus may promote neointima formation after percutaneous coronary interventions by facilitating cell cycle progression through inhibition of the eukariotic tumor suppressor protein p53. This prospective study sought to investigate the effect of previous cytomegalovirus infection on restenosis after coronary stenting. METHODS AND RESULTS: In 551 consecutive patients with successful stent placement, we determined cytomegalovirus IgG titers. Primary and secondary end points were the rate of angiographic restenosis at 6 months and the rate of target vessel reintervention at 1 year, respectively. Three hundred forty patients (62%) had a positive cytomegalovirus IgG titer. We obtained angiographic follow-up in 82% of all patients. Angiographic restenosis rate was 28.7% in patients with positive cytomegalovirus titers and 34.6% in patients with negative titers (P=0.18). Between the groups with and without positive cytomegalovirus titers, there were no significant differences in late lumen loss (1.16+/-0.90 mm and 1.23+/-0.86 mm, respectively, P=0.44). Target vessel reintervention was performed in 16.8% of the patients with positive cytomegalovirus titers and in 17.5% of those without (P=0.82). Even after correction for potential confounding variables by multivariate analysis, positive cytomegalovirus titers did not manifest as a predictor of angiographic restenosis (adjusted odds ratio [95% confidence interval], 0.78 [0.52 to 1.19]). CONCLUSIONS: Previous cytomegalovirus infection does not carry an increased risk of restenosis after stenting.

A transient assay for gene expression studies in B lymphocytes and its use for superantigen assays.

Superantigen assays are used to determine the stimulation of classes of T cells in response to the presence of a superantigen. This requires that antigen-presenting cells, such as B-lymphocytes, are co-cultured with syngeneic T cells over a four-day period. This prerequisite rules out conventional transient transfection procedures, since the cells must remain metabolically active for around five days. Consequently, stably transfected B cells have previously been used to test recombinant DNA constructs for superantigen activity. Here we present a protocol for the transient transfection of B cells that results in metabolically viable cells. Mouse mammary tumor virus (MMTV) encodes an endogenous superantigen. Using this transient transfection procedure, we show that a number of MMTV-based constructs give rise to functional superantigen activity. The ability to transiently transfect lymphocytes and maintain their viability should greatly facilitate studies of genes encoding products, such as superantigens, that can only be indirectly assayed on a second cell type as a result of expression in the recipient cell.

Crosslinked staphylococcal enterotoxin B stimulates CD8+ T cells only in the presence of unlinked costimulator signals.

The superantigen staphylococcal enterotoxin B (SEB) binds to class II MHC expressing cells and subsequently causes selective activation of T cells carrying appropriate T cell receptor (TCR) V beta chains. Apparently SEB acts as a bifunctional molecule by bridging class II MHC structures with the appropriate TCR-V beta chains. This assumption predicts that immobilized SEB ought to stimulate purified, class II MHC negative murine T cells. We show here that immobilized SEB lacks the ability to trigger murine CD8 T cells. Responsiveness obtained at a high T cell concentration is due to contaminating class II MHC-positive lymphocytes. Complementation of the culture system with syngeneic irradiated B cells blasts effectively restores responsiveness. The proliferating cells exhibit SEB specific cytotoxicity and a bias for V beta 8 expression. Since no evidence for leakiness of SEB covalently bound to sephadex beads was obtained, the data imply that immobilized SEB in fact binds to the TCR of T cells expressing the appropriate V beta chains. However, for primary activation additional costimulatory signals are required which can be provided in an unlinked fashion by activated B cells. Resting B cells are activated by immobilized SEB to cells expressing high costimulator activity. As such, the data point out a third function of SEB.

Superantigen mediated shock: a cytokine release syndrome.

Treatment of animals with superantigens results in profound immunological changes. A major fraction of all peripheral T cells becomes activated in vivo. Subsequently, successive waves of cytokines are produced with TNF playing a central pathophysiologic role. In addition, if the liver is damaged by an as yet poor defined mechanism the consequences of the cytokine syndrome are life threatening. However, TNF alone is not sufficient to cause death, instead synergizing interactions with cytokines like IL-1, IL-6, and IFN-gamma are probably involved. On the other hand, certain experimental conditions prevent these waves of cytokines and consequently lethal shock. Furthermore, a significant fraction of SA reactive T cells are deleted by programmed cell death 10 to 24 hours after treatment. Thereafter the surviving cells proliferate vigorously until day 2 or 3, followed by a second wave of apoptosis resulting in reduced SA reactive T cell numbers as compared to pretreatment levels. Of course, many aspects of the complicated events are only marginally understood and deserve further investigation.

Serum levels of end products of nitric oxide synthesis correlate positively with tumor necrosis factor alpha and negatively with body temperature in patients with postoperative abdominal sepsis.

Nitric oxide (NO) has been implicated as the principal mediator of the catecholamine resistant vasodilation in septic shock. In this pilot study, we wanted to know if the serum values of nitrite/nitrate (NO2/NO3), the stable endproducts of NO biosynthesis, are elevated in patients with septic shock. Furthermore, we investigated whether there is a correlation between NO2/NO3 serum levels and tumor necrosis factor alpha or interleukin 6. NO2/NO3 serum values were significantly elevated in septic patients compared to controls (72.1 +/- 6.1 vs. 35.7 +/- 9.2 microM, p < .001). There was a significant positive correlation between serum values of NO2/NO3 and tumor necrosis factor alpha (rs = 0.59, p < .001). In contrast, no correlation between NO2/NO3 and interleukin 6 was found. With the exception of body core temperature, which showed a negative correlation with NO2/NO3 levels, no clinical variable turned out to be significantly related to NO biosynthesis. These data indicate a potential role for NO in the clinical course of abdominal sepsis, but points out that more specific data has to be evaluated by prospective clinical studies in order to understand the complex pathophysiologic role of this novel mediator.

[Molecular diagnosis of mycobacterial infections]. / Molekulare Mykobakterien-Diagnostik.

Tuberculosis remains a leading cause of morbidity and mortality worldwide. A rapid and reliable diagnosis and discrimination from infections with nontuberculous mycobacteria (NTM) is critical. Frequently, formalin-fixed, paraffin-embedded (FFPE) tissues remain the only source for detection of micro-organisms in suspected cases of mycobacterial infection. Recently, numerous methods, including PCR assays, in situ hybridization and immunohistochemistry have been developed for detection of mycobacteria in FFPE samples. PCR-based assays are directed either against M.tbc.-specific sequences, such as IS6110, or amplify regions common to many mycobacterial species, e.g. the 65 kDa antigen, and then require sequencing or restriction fragment length polymorphism for species identification. Whereas the detection of DNA of M.tbc. in the correct setting is always of clinical relevance, the presence of various NTM species has to be interpreted with great caution due to their ubiquitous nature. However, the routine application of molecular tests has demonstrated that NTM infections are more common than previously thought, even in non-immunosuppressed hosts. The introduction of real-time PCR technology allows precise quantification of mycobacterial DNA and can be used for species identification through melting point analysis or appropriate DNA probes. Application of these assays originally developed for clinical microbiology offer a great opportunity for diagnostic improvement in molecular pathology as compared to qualitative PCR, mainly due to an increased specificity and a lower risk of contamination. Given the clinical impact of a positive molecular result for M. tbc., future efforts have to be aimed at standardization and quality control.

CpG-DNA upregulates the major acute-phase proteins SAA and SAP.

The acute-phase response is an immediate reaction of the host against invading microorganisms. We show here that oligodeoxynucleotides (ODNs) containing a CpG motif rapidly induce the major murine acute-phase proteins in vivo, i.e. serum amyloid A (SAA) and serum amyloid P (SAP). Serum levels of these proteins are elevated within 12 h and peak at 24 h after the injection of CpG-ODN or endotoxin. Liver cells produce the proteins with the same kinetics. Injection of interleukin 6 (IL-6), IL-1beta and tumour necrosis factor alpha (TNF-alpha) induces SAA and SAP in vivo, but the CpG-ODN-mediated induction does not depend on the presence of the TNF receptor p55, as the acute-phase response in TNF receptor p55-deficient mice does not differ from that of wild-type mice. Aside from CpG-ODN, bacterial genomic DNA also induces the acute-phase response in LPS-resistant C3H/Hej mice. The induction of the major acute-phase proteins SAA and SAP is blocked by the simultaneous injection of CpG-ODN together with D-galactosamine (D-GalN). As D-GalN sensitizes the host for the toxic effects of TNF-alpha, a possible mechanism could be the prevention of synthesis of the major acute-phase proteins SAA and SAP.