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The aim of this work was to establish an organ model for staphylococcal infection of human bone samples and to investigate the influence and efficacy of a microporous β-tricalcium phosphate ceramic (β-TCP, RMS Foundation) loaded with hydrogels (alginate, alginate-di-aldehyde (ADA)-gelatin) and clindamycin on infected human bone tissue over a period of 28 days. For this purpose, human tibia plateaus, collected during total knee replacement surgery, were used as a source of bone material. Samples were infected with S. aureus ATCC29213 and treated with differently loaded β-TCP composites (alginate +/- clindamycin, ADA-gelatin +/- clindamycin, unloaded). The loading of the composites was carried out by means of a flow chamber. The infection was observed for 28 days, quantifying bacteria in the medium and the osseus material on day 1, 7, 14, 21 and 28. All samples were histologically processed for bone vitality evaluation. Bone infection could be consistently performed within the organ model. In addition, a strong reduction in bacterial counts was recorded in the groups treated with ADA-gelatin + clindamycin and alginate + clindamycin, while the bacterial count in the control groups remained constant. No significant differences between groups could be observed in the number of lacunae filled with osteocytes suggesting no differences in bone vitality among groups. In an ex-vivo human bone infection model, over a period of 28 days bacterial growth could be reduced by treatment with ADA-Gel + CLI and ALG + CLI -releasing β-TCP composites. This could be relevant for its clinical use. Further work will be necessary to improve the loading of β-TCP and the bone infection organ model itself. STATEMENT OF SIGNIFICANCE: The common treatment of bone infections is debridement and systemic administration of antibiotics. In some cases, antibiotic-containing carriers are already used, but these must be removed again. Our work is intended to show another treatment option. The scaffold we have developed, made of a calcium phosphate ceramic and a hydrogel as the active substance carrier, can, in addition to releasing the active substance, also assume a load-bearing function of the bone and is biodegradable. In addition, the model we developed can also be used for the analysis and treatment of bone infections other than those of the musculoskeletal system. More importantly, it can also serve as a substitute for previously used animal experiments.

Item does not contain fulltext BACKGROUND: Since 2013, over 100 cases of Mycobacterium chimaera prosthetic valve endocarditis and disseminated disease were notified in Europe and the USA, linked to contaminated heater-cooler units (HCUs) used during cardiac surgery. We did a molecular epidemiological investigation to establish the source of these patients' disease. METHODS: We included 24 M chimaera isolates from 21 cardiac surgery-related patients in Switzerland, Germany, the Netherlands, and the UK, 218 M chimaera isolates from various types of HCUs in hospitals, from LivaNova (formerly Sorin; London, UK) and Maquet (Rastatt, Germany) brand HCU production sites, and unrelated environmental sources and patients, as well as eight Mycobacterium intracellulare isolates. Isolates were analysed by next-generation whole-genome sequencing using Illumina and Pacific Biosciences technologies, and compared with published M chimaera genomes. FINDINGS: Phylogenetic analysis based on whole-genome sequencing of 250 isolates revealed two major M chimaera groups. Cardiac surgery-related patient isolates were all classified into group 1, in which all, except one, formed a distinct subgroup. This subgroup also comprised isolates from 11 cardiac surgery-related patients reported from the USA, most isolates from LivaNova HCUs, and one from their production site. Isolates from other HCUs and unrelated patients were more widely distributed in the phylogenetic tree. INTERPRETATION: HCU contamination with M chimaera at the LivaNova factory seems a likely source for cardiothoracic surgery-related severe M chimaera infections diagnosed in Switzerland, Germany, the Netherlands, the UK, the USA, and Australia. Protective measures and heightened clinician awareness are essential to guarantee patient safety. FUNDING: Partly funded by the EU Horizon 2020 programme, its FP7 programme, the German Center for Infection Research (DZIF), the Swiss National Science Foundation, the Swiss Federal Office of Public Health, and National Institute of Health Research Oxford Health Protection Research Units on Healthcare Associated Infection and Antimicrobial Resistance.

We present an automated point-of-care testing (POCT) system for rapid detection of species- and resistance markers in methicillin-resistant Staphylococcus aureus (MRSA) at the level of single cells, directly from nasal swab samples. Our novel system allows clear differentiation between MRSA, methicillin-sensitive S. aureus (MSSA) and methicillin-resistant coagulase-negative staphylococci (MR-CoNS), which is not the case for currently used real-time quantitative PCR based systems. On top, the novel approach outcompetes the culture-based methods in terms of its short time-to-result (1 h vs. up to 60 h) and reduces manual labor. The walk-away test is fully automated on the centrifugal microfluidic LabDisk platform. The LabDisk cartridge comprises the unit operations swab-uptake, reagent pre-storage, distribution of the sample into 20 000 droplets, specific enzymatic lysis of Staphylococcus spp. and recombinase polymerase amplification (RPA) of species (vicK) - and resistance (mecA) -markers. LabDisk actuation, incubation and multi-channel fluorescence detection is demonstrated with a clinical isolate and spiked nasal swab samples down to a limit of detection (LOD) of 3 ± 0.3 CFU μl-1 for MRSA. The novel approach of the digital single cell detection is suggested to improve hospital admission screening, timely decision making, and goal-oriented antibiotic therapy. The implementation of a higher degree of multiplexing is required to translate the results into clinical practice.

Mycoplasma hominis is a rarely identified cause of respiratory infection that can cause significant morbidity and mortality in immunocompromised patients. It is often missed due to the difficult detection of the organism with routine laboratory methods. We present the case of a 63-year-old male with a history of lymphoma who was transferred to our hospital with recurrent right-sided empyema and lung abscess in the right lower lobe. Advanced microbiological analysis finally revealed infection with M hominis. Despite appropriate antibiotic treatment, prolonged drainage as well as repeated surgery, which eventually resulted in right lower bilobectomy, were necessary for clinical improvement of our patient. Infection with M hominis may be more prevalent than previously indicated and can cause severe morbidity and mortality in thoracic surgery patients. Due to the diagnostic challenge, the appropriate antimicrobial treatment is often delayed. Inherent resistance to macrolides and inactivity of cell wall-active agents potentially complicate empiric antibiotic therapy. A review of the currently available literature enables a better understanding of the diagnostic difficulties and importance of this infection.

The aim of this work was to establish an organ model for staphylococcal infection of human bone samples and to investigate the influence and efficacy of a microporous β-tricalcium phosphate ceramic (β-TCP, RMS Foundation) loaded with hydrogels (alginate, alginate-di-aldehyde (ADA)-gelatin) and clindamycin on infected human bone tissue over a period of 28 days. For this purpose, human tibia plateaus, collected during total knee replacement surgery, were used as a source of bone material. Samples were infected with S. aureus ATCC29213 and treated with differently loaded β-TCP composites (alginate +/- clindamycin, ADA-gelatin +/- clindamycin, unloaded). The loading of the composites was carried out by means of a flow chamber. The infection was observed for 28 days, quantifying bacteria in the medium and the osseus material on day 1, 7, 14, 21 and 28. All samples were histologically processed for bone vitality evaluation. Bone infection could be consistently performed within the organ model. In addition, a strong reduction in bacterial counts was recorded in the groups treated with ADA-gelatin + clindamycin and alginate + clindamycin, while the bacterial count in the control groups remained constant. No significant differences between groups could be observed in the number of lacunae filled with osteocytes suggesting no differences in bone vitality among groups.

Quantitative PCR (qPCR) assays are the gold standard for diagnosis of Pneumocystis jirovecii pneumonia (PCP). However, they are laborious and require skilled personnel. Therefore, execution outside regular working hours of the molecular biology laboratory is limited. The eazyplex P. jirovecii assay (PJA) uses loop-mediated isothermal amplification for detection of P. jirovecii. It is performed directly with respiratory specimens, without the need for special skills, and delivers a result within 3 to 25 min. The goal of our study was to compare the performance of the eazyplex PJA with that of established P. jirovecii qPCR assays. All archived bronchoalveolar lavage fluid (BALF) samples that had previously tested positive for P. jirovecii by qPCR assay and 50 control samples (retrospective part), as well as all BALF samples received for P. jirovecii analysis over a period of 4 months (prospective part), were tested. Forty-nine patients with proven PCP and 126 patients without PCP were included. The sensitivity and specificity of the eazyplex PJA (95.7% and 96.5%, respectively) were comparable to those for three different P. jirovecii qPCR assays. The detection limit of the eazyplex PJA was analogous to 103 copies of the major surface glycoprotein gene per 25 μl of BALF, corresponding to 10 to 20 P. jirovecii cells. The eazyplex PJA reliably discriminated patients with PCP from patients with P. jirovecii colonization. It delivered a positive result within a mean of 9 min 38 s and required a hands-on time of 2 min 45 s. In summary, the eazyplex PJA showed identical performance for the diagnosis of PCP, compared to qPCR assays. However, in terms of time to result, practicability, and robustness, the eazyplex PJA is clearly superior and allows for around-the-clock molecular testing.

Summary Background Several cases of Mycobacterium chimaera infection have recently been reported in cardiosurgical patients. So-called heater–cooler units (HCUs) used in cardiosurgical procedures are suspected to be the reservoir for pathogen growth and dissemination. Aim To assess the contamination status of HCUs at our facility. Methods Air sampling for mycobacteria was conducted at different distances from the machines and in the area around the operating table. Air sampling was also conducted for non-fermenters as a surrogate parameter for water-associated pathogens. Findings Mycobacterium chimaera was detected in the water tanks of the HCUs. When the devices were operating, M. chimaera was also found in their exhaust air, as well as in the area around the operating table. Non-fermenters were identified at different distances from the running HCU and the area around the operating table. Cultures remained negative when the devices were switched off. Conclusions Exhaust air from HCUs may be a pathway of pathogen transmission to patients undergoing open chest heart surgery. Although, for technical reasons, relocation of HCUs is difficult to achieve, only strict separation of the HCU from the operating room appears to enhance patient safety. Using non-fermenters as a surrogate parameter may be considered a viable option for a timely risk assessment. The design of HCUs should be modified to keep susceptibility to contamination at a minimum.

Objective The aim of this study was to produce a novel composite of microporous β-TCP filled with alginate and Vancomycin (VAN) to prolong the release behavior of the antibiotic for up to 28 days. Material and methods Using the flow chamber developed by the group, porous ceramics in a directional flow were filled with alginates of different composition containing 50 mg/mL of antibiotics. After cross-linking the alginate with calcium ions, incubation took place in 10 mL double-distilled water for 4 weeks at 37 °C. At defined times (1, 2, 3, 6, 9, 14, 20 and 28 days), the liquid was completely exchanged and analyzed by capillary zone electrophoresis and microtiter trials. For statistical purposes, the mean and standard deviation were calculated and analyzed by ANOVA. Results The release of VAN from alginate was carried out via an external calcium source over the entire period with concentrations above the minimal inhibitory concentration (MIC). The burst release measured 35.2 ± 1.5%. The release of VAN from alginate with an internal calcium source could only be observed over 14 days. The burst release here was 61.9 ± 4.3%. The native alginate’s burst release was 54.1 ± 7.8%; that of the sterile alginate 40.5 ± 6.4%. The microtiter experiments revealed efficacy over the entire study period for VAN. The MIC value was determined in the release experiments as well in a range of 0.5–2.0 μg/mL against Staphylococcus aureus . Statement of Significance Drug release systems based on β-TCP and hydrogels are well documented in literature. However, in all described systems the ceramic, as granule or powder, is inserted into a hydrogel. In our work, we do the opposite, a hydrogel which acts as reservoir for antibiotics is placed into a porous biodegradable ceramic. Eventually, this system should be applied as treatment of bone infections. Contrary to the “granule in hydrogel” composites it has the advantage of mechanical stability. Thus, it can take over functions of the bone during the healing process. For a quicker translation from our scientific research into clinical use, only FDA approved materials were used in this work.

Mycoplasma salivarium is a rare agent of septic arthritis in immunocompromised patients. We report a case of septic arthritis due to Mycoplasma salivarium in a patient with B-cell chronic lymphocytic leukemia who underwent chemotherapy with rituximab and bendamustin. Therapy of arthritis due to Mycoplasma salivarium is difficult because there are almost no susceptibility data available. The present case illustrates that antimicrobial susceptibility of Mycoplasma strains is not necessarily predictable and that antibiotic therapy should therefore be guided by in vitro susceptibility testing.

Pneumocystis jirovecii (carinii) pneumonia (PJP) is a major cause of disease in immunocompromised individuals. However, until recently no reliable and specific serological parameters for the diagnosis of PJP have been available. (1 → 3)-β-d-Glucan (BG) is a cell wall component of P. jirovecii and of various other fungi. Data from the past few years have pointed to serum measurement of BG as a promising new tool for the diagnosis of PJP. We therefore conducted a retrospective study on 50 patients with PJP and 50 immunocompromised control patients to evaluate the diagnostic performance of serum BG measurement. Our results show an excellent diagnostic performance with a sensitivity of 98.0% and a specificity of 94%. While the positive predictive value was only 64.7%, the negative predictive value was 99.8% and therefore a negative BG result almost rules out PJP. BG levels were already strongly elevated in an average of 5 days and up to 21 days before microbiological diagnosis demonstrating that the diagnosis could have been confirmed earlier. BG levels at diagnosis and maximum BG levels during follow-up did not correlate with the outcome of patients or with the P. jirovecii burden in the lung as detected by Real-Time PCR. Therefore, absolute BG levels seem to be of no prognostic value. Altogether, BG is a reliable parameter for the diagnosis of PJP and could be used as a preliminary test for patients at risk before a bronchoalveolar lavage is performed.

Although tularemia is a long-known disease, its significance had diminished over the last decades in Middle Europe. However, over the past years, there is new evidence suggesting that tularemia has re-emerged in Germany. In 2007, the highest number of human cases for almost 50 years has been notified. Beside typical vectors, new ways of transmission seem to gain significance. So far, mosquito bite-transmitted tularemia has only been known from Scandinavia but not from Middle Europe.We report the first case of a 1-year-old toddler from Southwestern Germany with mosquito bite-associated ulceroglandular tularaemia. The new and interesting features of this case are the young age of the patient and the unusual transmission route. The available data suggesting changes in the epidemiology for tularemia in Germany are reviewed. This is an interesting case of infantile tularemia with a very unusual transmission route, highlighting ongoing changes in the epidemiology of tularemia in Germany.

Background Multidrug-resistant Escherichia coli and other enteric bacteria producing extended-spectrum β-lactamases (ESBL) have emerged as an important cause of invasive infection. Targeting the primary (intestinal) niche by decolonization may be a valuable approach to decrease the risk of relapsing infections and to reduce transmission of ESBL-producing enteric pathogens. Methods In a retrospective observational study we evaluated the efficacy of intestinal decolonization treatment using orally administered colistin or other non-absorbable agents given for 2 to 4 weeks in adult patients with previous relapsing infection and persistent intestinal colonization with ESBL-positive Enterobacteriaceae (ESBL-E). Eradication success was defined as negative rectal swab or stool culture at the end of treatment and at follow up-2 weeks after treatment discontinuation. Results First-line decolonization treatment led to eradication of ESBL-E in 19/45 patients (42 %, 7/18 low-dose [4 × 1 million units] colistin, 3/12 high-dose [4 × 2 million units] colistin, 9/15 rifaximin [2 × 400 mg]), and secondary/salvage treatment was successful in 8/13 patients (62 %, 20 treatment episodes). Late follow-up showed that 7/13 patients (54 %) with successful initial or salvage decolonization became recolonized within 3 months after post-treatment assessment while all eight of the patients failing initial or salvage decolonization treatment with late follow-up remained colonized. A narrative review of the literature confirms the limited efficacy of non-absorbable antibiotics including conventional selective digestive tract decolonization (SDD)-like combination regimens for eradicating multidrug-resistant enteric bacteria from the intestinal tract. Conclusions At present, there is no clear evidence of a significant decolonization efficacy using single-drug treatment with oral non-absorbable antibiotics. More effective regimens are needed and a better definition of at risk patients is required for planning meaningful randomized controlled studies in this field.

Aims We identified 10 patients with disseminated Mycobacterium chimaera infections subsequent to open-heart surgery at three European Hospitals. Infections originated from the heater–cooler unit of the heart–lung machine. Here we describe clinical aspects and treatment course of this novel clinical entity. Methods and results Interdisciplinary care and follow-up of all patients was documented by the study team. Patients’ characteristics, clinical manifestations, microbiological findings, and therapeutic measures including surgical reinterventions were reviewed and treatment outcomes are described. The 10 patients comprise a 1-year-old child and nine adults with a median age of 61 years (range 36–76 years). The median duration from cardiac surgery to diagnosis was 21 (range 5–40) months. All patients had prosthetic material-associated infections with either prosthetic valve endocarditis, aortic graft infection, myocarditis, or infection of the prosthetic material following banding of the pulmonary artery. Extracardiac manifestations preceded cardiovascular disease in some cases. Despite targeted antimicrobial therapy, M. chimaera infection required cardiosurgical reinterventions in eight patients. Six out of 10 patients experienced breakthrough infections, of which four were fatal. Three patients are in a post-treatment monitoring period. Conclusion Healthcare-associated infections due to M. chimaera occurred in patients subsequent to cardiac surgery with extracorporeal circulation and implantation of prosthetic material. Infections became clinically apparent after a time lag of months to years. Mycobacterium chimaera infections are easily missed by routine bacterial diagnostics and outcome is poor despite long-term antimycobacterial therapy, probably because biofilm formation hinders eradication of pathogens.

The incidence of vancomycin-resistant Enterococcus faecium isolation was low (

To investigate the question what happens to the tissue lost at the entrance wound, experimental studies were performed on composite models consisting of dyed pig skin and gelatin blocks. For the test shots to the skin-gelatin preparations, cartridges calibre .38 spec. with different bullet types (round nose, hollow point, flat nose, truncated cone) were used. In all shots, a multitude of coloured skin particles were macroscopically discernible along the bullet tracks. In addition, small cell aggregations could be demonstrated microscopically even in those sections of the bullet paths which did not show skin fragments visible to the naked eye. The distribution of the skin particles showed certain peculiarities depending on the type of projectile.

Objectives - Tuberculous meningoencephalitis (TBM), an infrequent disease in Western European countries, shows a wide heterogeneity of clinical symptoms. Material and methods We present 4 patients (age range 42- 72 years) with the definite diagnosis of isolated TBM. All patients were HIV-seronegative, only I patient was known to be immunoincompetent on admission due to acute myelocytic leukemia; other reasons for immune suppression were detected in 2 other patients (leukemia and idiopathic CD4 + T-lymphocytopenia, respectively). Results The diagnosis of TBM was confirmed in 3 cases by culture from CSF, in 1 case Mycobacterium tuberculosis was proven only in tracheal aspirate. In I patient M. bovis was found, which is an extremely rare cause of TBM in Germany. We report the contributions of different diagnostic tools (CSF analysis, neuroimaging) in reaching the presumptive diagnosis and in monitoring the further course. All patients developed neurological complications despite prompt tuberculostatic treatment. Three of the patients presented a chronic severe loss of consciousness of unclear origin. Conclusion The possible causative relationships of these complications and their impact on the prognosis are discussed.

Since the widespread availability and use of antibiotics the prevalence of Lemierre syndrome (L.S.) has decreased. It is a well-described entity, consisting of postanginal septicaemia with thrombophlebitis of the internal jugular vein with metastatic infection, most commonly in the lungs. The most common causative agent is a gram-negative, non-spore-forming obligate anaerobic bacterium, Fusobacterium necrophorum (F.n.). We describe the unusual clinical features of a 12-year-old boy with Lemierre syndrome with isolated hypoglossal nerve palsy - the latter symptom is an extremely rare manifestation of this disease.

Mucorales (subphylum Mucoromycotina ) are well-known agents of invasive mucormycosis, whereas Entomophthorales (subphylum Entomophthoromycotina ) are rarely encountered in human diseases in temperate zones. Here we report a fatal case of invasive rhino-orbitocerebral entomophthoramycosis caused by Conidiobolus incongruus in a 78-year-old woman with myelodysplastic syndrome.

Paenibacillus larvae causes American foulbrood in honey bees. We describe P. larvae bacteremia in 5 injection drug users who had self-injected honey-prepared methadone proven to contain P. larvae spores. That such preparations may be contaminated with spores of this organism is not well known among pharmacists, physicians, and addicts.

The aim of this study was to isolate and detect microorganisms of root-filled teeth associated with periradicular lesions. Specimens were sampled from patients undergoing root canal retreatment. The bacteria were characterized by morphologic and biochemical analysis and by 16S rRNA gene sequencing. Microorganisms were detected in 10 of 18 teeth. The majority of positive samples revealed a mixed culture of 2-8 species. In 2 teeth Enterococcus faecalis was the only detected species. For the first time Vagococcus fluvialis was detected in root canals. Solobacterium moorei and Fusobacterium nucleatum were the most prevalent species. Presence of F. nucleatum was associated with the presence of S. moorei in 5 of 7 cases. In all teeth with Parvimonas micra and Dialister invisus, F. nucleatum and S. moorei were found. Moreover, members of additional different genera were detected delivering bacterial compositions that have been not described yet.

Background Ethyl glucuronide (EtG) and ethyl sulphate (EtS) are specific and sensitive markers of ethanol consumption well established in monitoring withdrawal treatment in patients with chronic alcoholism. Recently, bacterial decomposition as well as in vitro and post-mortem formation of EtG was reported. The aim of this study was to investigate the influence of different preservatives on the stability of EtG and EtS concentrations in urine samples. Methods Urine samples were doped with glucuronidase-positive Escherichia coli after sterile filtration. The preservatives used were thymol, chlorhexidine, boric acid and the combination of chlorhexidine, ethylparabene and sodium propionate. Different aliquots of urine samples were stored refrigerated (4–8 °C), at room temperature (18 ± 1 °C) and in an incubator (36 ± 1 °C) for a period of 9 days with daily sampling. EtG and EtS analyses were performed by LC–ESI-MS/MS. The number of bacteria was detected by counting the colony forming units on Columbia blood agar plates. Results and conclusions Chlorhexidine on its own as well as in the aforementioned combination, and boric acid proved useful preservatives, while EtG degraded in samples doped with thymol. Addition of these preservatives did not interfere with the LC–MS/MS analysis.

Recent studies show that ethyl glucuronide (EtG) can be decomposed by bacteria; whilst so far no degradation of ethyl sulphate (EtS) has been observed. In the present study, in vitro experiments with bacterial colonies were performed. Bacteria (Escherichia coli, Klebsiella pneumoniae, Clostridium sordellii) were isolated from autopsy material (liver, heart blood, urine, ascites, pericardial fluid, pleural fluid) tested for beta-glucuronidase activity, and three bacterial strains were added to nutrient-deficient medium containing EtG and/or EtS and incubated at 36 +/- 1 degrees C. Samples were taken after various intervals up to 11 days, and EtG and EtS were determined by electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS). EtG was degraded by E. coli and C. sordellii--complete degradation occurred in the range of 3-4 days--and these bacteria exhibited beta-glucuronidase activity. EtS was not affected within 11 days of incubation.

Although the prevalence of carbapenemase-producing Acinetobacter baumannii (CP-AB) in Germany is still low an increasing number of outbreaks has been reported in recent years. In August 2014 we observed a cluster of 4 patients with CP-AB (OXA-23) on our surgical intensive care unit (SICU).

To the Editor: Advanced molecular biologic methods have improved the species differentiation and taxonomic classification of microorganisms, including nontuberculous mycobacteria. Identifying and characterizing an increasing number of "new" mycobacterial species of medical importance is now possible. Often, these newly described mycobacteria have been isolated from immunocompromised patients (1,2). Some of those have been difficult to cultivate, and 16S rRNA gene sequencing or similar methods have become of major importance to allow species identification and clinical diagnosis. Here, we report 2 patients with disease likely caused by a novel mycobacterial species that could not been previously cultivated. Diagnosis relied on molecular identification of acid-fast organisms in tissues. We also briefly review 2 similar cases published previously and note that all 4 known patients were from central Europe. A 43-year-old woman without evidence of immunodeficiency reported recurrent episodes of dysuria, hematuria, and abdominal discomfort for >1 year. On cystoscopy, a hyperemic bladder with yellow plaques was observed. Biopsy of the plaques showed granulomatous infiltration of histiocytes. No definite diagnosis was made, and symptomatic relief occurred after a trial of empiric antimicrobial drug therapy. When the patient sought treatment again with persistent abdominal discomfort, endoscopy showed a lesion in the stomach that resembled a healed ulcer and numerous elevated yellow plaques throughout the colon and ileum. Microscopically, a granulomatous inflammation with macrophages filled with many acid-fast rods was seen, but mycobacterial growth did not occur in different media or in a guinea pig. Antituberculous treatment was initiated, and the patient slowly improved. A repeat colonoscopy showed fewer and smaller lesions. Efforts to culture the organism from biopsy specimens were again unsuccessful (different solid and liquid media, blood or chocolate agar, guinea pig, Balb/c mice). Sequencing of polymerase chain reaction (PCR) products of the 16S rRNA gene (3) from the organism represented a previously unknown mycobacterial species (EMBL DNA database: accession number {"type":"entrez-nucleotide","attrs":{"text":"Z50172","term_id":"1480398","term_text":"Z50172"}}Z50172). "Mycobacterium tilburgii" was proposed as the designation of this species because the novel mycobacterium was identified in the city of Tilburg (4). Retrospective analysis of the initial bladder biopsy specimen and of 2 lymph nodes taken during abdominal surgery (which became necessary because of a complicating ileal obstruction) confirmed the presence of "M. tilburgii" 16S rRNA gene sequences. All samples yielding PCR fragments hybridized with a "M. tilburgii"–specific biotinylated DNA probe. A 34-year-old AIDS patient sought treatment for involuntary weight loss. Endoscopy showed white superficial embossments of the duodenal mucosa. Biopsy specimens were negative for acid-fast bacteria and mycobacterial growth (again, different solid and liquid media, extended incubation periods). Antiretroviral therapy was begun, but asthenia, persistent fever, diarrhea, vomiting, and cachexia developed. Repeat endoscopy showed yellow, plaquelike lesions in the duodenum (Figure A1) and esophagus with periodic acid–Schiff (PAS) and acid-fast intracellular bacteria that were nonculturable. PCR of 16S rRNA gene (3) confirmed the presence of mycobacterial DNA; sequencing showed 100% homology to the species with the proposed name "M. tilburgii" (4). Retrospectively, "M. tilburgii"16S rRNA was present in the initial duodenal biopsy specimen, when no mycobacteria were detected microscopically. Combination therapy led to a gradual disappearance of fever and diarrhea. The patient's weight increased, and the lesions disappeared (Table). Control endoscopy results were unremarkable, although duodenal biopsy specimens showed PAS-positive material. Mycobacterial DNA was no longer detectable. Table Characteristics of patients with confirmed "Mycobacterium tilburgii" disease* We believe that the identified acid-fast organism named "M. tilburgii" was the causative agent of illness in the 2 patients. First, the clinical and histopathologic appearance of the lesions is compatible with mycobacterial infection. Second, the finding that the macrophages in these lesions contained large numbers of acid-fast bacteria supports the conclusion that mycobacterial infection caused the lesion. Third, the involvement of normally sterile locations such as lymph nodes, and the improvement after antimycobacterial therapy with disappearance of the lesions, acid-fast organisms, and mycobacterial DNA supports a causative role of the organism rather than a bystander role. Attempts to culture the organism from biopsy specimens and resection materials remained unsuccessful, although specific requirements for organisms like M. genavense, a mycobacterium that also is not cultivable on regular mycobacterial media (5), were met. Diagnosis therefore had to rely on sequencing of PCR products. Based on 16S rRNA homology, "M. tilburgii" shows close relationship to M. sherrisii, a M. simiae–related mycobacterium (6) that probably corresponds to Mycobacterium strain IWGMT 90143 (accession no. {"type":"entrez-nucleotide","attrs":{"text":"X88906","term_id":"1237223","term_text":"X88906"}}X88906) (7). "M. tilburgii" also has a high homology to Mycobacterium sp. Murphy, a mycobacterial species that is considered the cause of canine lepra and that also has not been cultivated so far (8) (accession no. {"type":"entrez-nucleotide","attrs":{"text":"AF144747","term_id":"4769067","term_text":"AF144747"}}AF144747). These 2 patients double the number of patients reported previously (Table). Both previously reported M. tilburgii patients had AIDS, 1 of whom had a disease similar to that of the second patient in this report (9). Taken together, 3 patients had gastrointestinal involvement with yellow mucosal plaques (Figure A1), and 1 patient had pulmonary nodules (10). One of the 4 patients did not have a detectable immunodeficiency, yet had gastrointestinal and urinary bladder lesions. Although complications occurred, treatment was successful in all 4 cases with usual anti–Mycobacterium avium complex therapy. Interestingly, all 4 patients so far are of middle-European descent. This species may be geographically confined, similar to the occurrence of M. malmoense in which most clinical isolates come from northern Europe (1).

The skin defect of a gunshot entrance wound is caused by the retrograde and anterograde displacement of skin particles. In the present study, we investigated whether gunshots to bacterially contaminated skin are associated with the transport of micro-organisms into the bullet path. The shots were fired into composite models of pig skin and gelatin blocks. The outer surface of the skin was covered with a thin layer of a defined bacterial suspension [green fluorescent protein-labelled Escherichia coli in the preliminary test and Staphylococcus epidermidis, DSM 1798, in the main test series]. After the bacterially contaminated fluid had dried, test shots were fired from a distance of 5 and 10 m using calibre .38 Special cartridges with different bullet types (round nose, truncated cone, hollow point and flat nose). Subsequent bacteriological analyses showed that all the bullet tracks in the gelatin serving as tissue simulant contained displaced micro-organisms from the skin surface. The results are presented and discussed with reference to the transport of skin particles into the depth of the wound track.

The authors report on the case of a 18-year-old student who died suddenly and unexpectedly of a pericardial tamponade after perforated ulcer-polypous endocarditis of the aortic valve. The detection of aerococcus urinae--which usually causes urinary tract infection--on the inflamed aortic valve demonstrates an unusual pathogenetic chain linking complexes of findings in the urogenital system and the heart.

Tuberculous meningoencephalitis (TBM) is still associated with a high mortality. The relative rareness of TBM in Western European countries and the accompanying heterogeneous and unspecific clinical symptoms often result in a delayed diagnosis.We present six HIV-negative patients (age 37-72 years) with a laboratory-confirmed or clinically probable diagnosis of TBM. The diagnosis could be confirmed in three patients by culture of the cerebrospinal fluid (CSF), in one patient by positive tracheal aspirate culture. In the cases with probable TBM, the diagnosis was confirmed by the combination of clinical symptoms, CSF analysis, and magnetic resonance imaging (MRI).The diagnostic and therapeutic problems in TBM are discussed. Moreover, the neurologic complications are presented which developed in all patients during the clinical course despite immediate antituberculous therapy.

Rifampin in combination with erythromycin is a recommended treatment for severe cases of legionellosis. Mutations in the rpoB gene are known to cause rifampin resistance in Escherichia coli and Mycobacterium tuberculosis, and the purpose of the present study was to investigate a possible similar resistance mechanism within the members of the family Legionellaceae. Since the RNA polymerase genes of this genus have never been characterized, the DNA sequence of the Legionella pneumophila rpoB gene was determined by the Vectorette technique for genome walking. A 4,647-bp DNA sequence that contained the open reading frame (ORF) of the rpoB gene (4,104 bp) and an ORF of 384 bp representing part of the rpoC gene was obtained. A 316-bp DNA fragment in the center of the L. pneumophila rpoB gene, corresponding to a previously described site for mutations leading to rifampin resistance in M. tuberculosis, was sequenced from 18 rifampin-resistant Legionella isolates representing four species (L. bozemanii, L. longbeachae, L. micdadei, and L. pneumophila), and the sequences were compared to the sequences of the fragments from the parent (rifampin-sensitive) strains. Six single-base mutations which led to amino acid substitutions at five different positions were identified. A single strain did not contain any mutations in the 316-bp fragment. This study represents the characterization of a hitherto undescribed resistance mechanism within the family Legionellaceae.