Alexander Mischnik

Integration of matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry in blood culture diagnostics: a fast and effective approach

Sepsis is a major cause of mortality in hospitalized patients worldwide, with lethality rates ranging from 30 to 70 %. Sepsis is caused by a variety of different pathogens, and rapid diagnosis is of outstanding importance, as early and adequate antimicrobial therapy correlates with positive clinical outcome. In recent years, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) fingerprinting has become a powerful tool in microbiological diagnostics. The direct identification of micro-organisms in a positive blood culture by MALDI-TOF MS can shorten the diagnostic procedure significantly. Therefore, the aim of the present study was to evaluate whether identification rates could be improved by using the new Sepsityper kit from Bruker Daltonics for direct isolation and identification of bacteria from positive blood cultures by MALDI-TOF MS compared with the use of conventional separator gel columns, and to integrate the MALDI-TOF MS-based identification method into the routine course of blood culture diagnostics in the setting of a microbiological laboratory at a university hospital in Germany. The identification of Gram-negative bacteria by MALDI-TOF MS was significantly better using the Sepsityper kit compared with a separator gel tube-based method (99 and 68 % correct identification, respectively). For Gram-positive bacteria, only 73 % were correctly identified by MALDI-TOF with the Sepsityper kit and 59 % with the separator gel tube assay. A major problem of both methods was the poor identification of Gram-positive grape-like clustered cocci. As differentiation of Staphylococcus aureus from coagulase-negative staphylococci is of clinical importance, a PCR was additionally established that was capable of identifying S. aureus directly from positive blood cultures, thus closing this diagnostic gap. Another benefit of the PCR approach is the possibility of directly detecting the genes responsible for meticillin resistance in staphylococci and for vancomycin resistance in enterococci, which is of high importance for early adequate treatment. Both of the described methods were finally integrated into a protocol for fast and effective identification of bacteria from positive blood cultures.

Microbiology and resistance in first episodes of spontaneous bacterial peritonitis: implications for management and prognosis.

International guidelines for antibiotic treatment of spontaneous bacterial peritonitis (SBP) are based on studies conducted decades ago and do not reflect regional differences of bacterial epidemiology.

Relapse of posttraumatic osteomyelitis due to Clostridium celerecrescens.

In October 2009, a 55-year-old male with diabetes mellitus, probably since 2007, peripheral artery occlusive disease (PAOD) without any notion of peripheral neuropathy, and a history of alcoholism in 2003 presented with right lower leg pain accompanied by local hyperthermia, swelling, and reddening without fever. The medical history revealed that, in August 2000, the patient had suffered from a grade III open comminuted tibia fracture associated with a compartment syndrome. The fracture had been fixed using intramedullary nailing and fasciotomy at the lower leg had been performed. An intraoperative swab had yielded aerobic spore-forming bacteria after enrichment culture without further differentiation, but no anaerobic bacteria. In August 2001, the patient presented with a pus-filled fistula at the distal third of the lower leg. At this time point, the intramedullary nail was removed and the tibia was reamed for the treatment of osteomyelitis. Staphylococcus aureus (S. aureus) was grown from intraoperative specimens with resistance to penicillin and ampicillin only. Intravenous cefuroxime was given for 34 days. After removal of the implant, including reaming of the long bone and aggressive debridement, the fistula at the lower leg healed slowly. In the following 8 years, no event of trauma, manipulation, or surgery to the right lower leg was recorded. Upon readmission, plain radiographs of the right lower leg showed the radiological sequelae of comminuted lower leg fracture with full consolidation of the fracture lines. Typical signal alterations were found at magnetic resonance imaging (MRI) scanning within the medullary canal, including enhancement after the application of contrast agent, suggesting osteomyelitis within the distal third of the tibia. The tibia was opened proximally and a phlegmon of the medullary cavity was drained. Aggressive wound debridement, including reaming and jet lavage, was done. A rod was formed from polymethylmethacrylate (PMMA) bone cement and introduced as a local drug delivery system for gentamicin. Intraoperative swab of the right tibia and tissue specimens taken from the medullary cavity tissue revealed a Gram-positive anaerobic rod. The bacterium was identified as Clostridium clostridioforme (C. clostridioforme) at 99.8% using API rapid 32 A strips (bioMerieux, France). In contrast, matrix-assisted laser desorption/ionization timeof-flight (MALDI-TOF) identification detected C. sphenoides with an identification score of 1.6. For exact species determination, 16S rRNA gene sequence comparisons were performed (GATC, Germany), and the isolated bacterium was identified as C. celerecrescens (99% identity from a 512-nt fragment). Antibiotic susceptibilities were determined by Etest (bioMerieux, France). The bacillus was susceptible to amoxicillin/clavulanic acid (minimum inhibitory concentration [MIC] 2 mg/L), clindamycin (MIC 2 mg/L), imipenem (MIC 2 mg/L), and metronidazole (MIC 0.06 mg/L). To provide broad coverage, a treatment including cefuroxime (1,500 mg three times/day) plus metronidazole (400 mg three times/day) with intravenous administration for 15 days was begun. Twelve days later, the tibia was revised. The bone cement rod was removed and repetitive surgical debridement, including A. Mischnik (&) S. Zimmermann I. Bekeredjian-Ding Department of Infectious Diseases, University of Heidelberg, Medical Microbiology and Hygiene, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany e-mail: alexander.mischnik@med.uni-heidelberg.de

False non-susceptible results of tigecycline susceptibility testing against Enterobacteriaceae by an automated system: a multicentre study.

It has been previously shown that different antimicrobial susceptibility testing (AST) methodologies can influence susceptibility results for tigecycline (Hope et al., 2010; Cohen Stuart et al., 2010; Marchaim et al., 2014; Torrico et al., 2010). Even within a single AST method, several testing conditions including medium age and ion content may cause discrepant results (Bradford et al., 2005; FernandezMazarrasa et al., 2009). For tigecycline, Leal Castro et al. (2010) and Lat et al. (2011) provided early data demonstrating overestimation of MICs and resistance rates by Vitek 2 system. Our study, therefore, aimed to systematically determine the error rates of tigecycline AST for Enterobacteriaceae by Vitek 2 (focusing on false non-susceptible results) as well as other automated systems in comparison to a reference method. Additionally, we compared several other AST methods and testing conditions.

Salmonella enterica serovar Minnesota urosepsis in a patient with Crohn's disease in the absence of recent or current gastrointestinal symptoms.

Salmonella enterica serovar Minnesota is a rarely isolated organism in clinical samples mainly grown from stool cultures. Sepsis due to Salmonella is known in severely immunocompromised patients, but so far urosepsis due to S. enterica serovar Minnesota has not been described. We report a case of a 31-year-old patient suffering from Crohns disease treated with infliximab and azathioprine, in whom was implanted a double-J ureteric catheter for urolithiasis. The patient presented with urinary tract infection and severe sepsis. S. enterica serovar Minnesota was grown from urine and blood cultures. After empiric antimicrobial treatment with meropenem and vancomycin, treatment was changed to ceftriaxone. Antimicrobial treatment was continued for a total of 3 weeks without evidence of Salmonella recurrence on follow-up visits. Salmonella spp. rarely cause urinary tract infection and sepsis. However, in immunocompromised patients, non-typhoidal salmonellosis merits a thorough clinical and microbiological evaluation.

Haemophilia-associated Yersinia pseudotuberculosis serotype O:1 septicaemia: the role of iron

Septicaemia and septic arthritis due to Yersinia pseudotuberculosis are rare diseases with high mortality rates. Reactive arthritis caused by Yersinia infection is a well-known complication but septic arthritis is found at a much lower frequency. It has already been established that there is a relationship between yersiniosis and iron but there are currently no data about yersiniosis and haematological disorders such as haemophilia. We report a case of septic arthritis due to Y. pseudotuberculosis as an early manifestation of Yersinia septicaemia in a patient with severe haemophilia A. The patient had no history of immunosuppression and presented with a repeat case of haemarthrosis with a fever of unknown origin. Furthermore, he suffered from acute-on-chronic renal failure and non-ST segment elevation myocardial infarction. Arthrocentesis and blood culture tested positive for Y. pseudotuberculosis. Iron deposits at localized sites in the synovium in patients with haemophilia have been described, and as Yersinia infections are common in patients with secondary iron overload, we felt that a review of the literature was in order. Severe Yersinia infection is often associated with iron overload, a condition that might occur as a side effect in the treatment of haemophilia. Iron overload, which plays an important role in the pathogenesis of haemophilic arthropathy, may have increased the virulence of Y. pseudotuberculosis in the present case.

First case of disseminated cryptococcosis in a Gorilla gorilla.

In humans, Cryptococcus mainly infects individuals with HIV infection or other types of immunosuppression. Here, we report the first case of disseminated cryptococcosis in a simian immunodeficiency virus‐negative 27‐year‐old female Gorilla gorilla presenting with lethargy, progressive weight loss and productive cough. The diagnosis was confirmed by positive lung biopsy culture, serum cryptococcal antigen, and cerebral histopathology demonstrating encapsulated yeasts. Molecular characterisation of lung culture isolate yielded Cryptococcus neoformans var. grubii. An immune‐deficiency could not be demonstrated.

A persistent case of tuberculosis of the pubic symphysis and pubic bone.

A case of a patient suffering from tuberculosis of the pubic symphysis and pubic bone is presented. There were no symptoms other than pain in the right groin area for 12 months. An X-ray of the pelvis showed an osteodestructive lesion of the pubic bone, and an MRI revealed an abscess formation of the pubic symphysis. Tissue samples were collected via CT-guided needle biopsy. Histological evaluation of tissue and analysis by PCR prompted the diagnosis of musculoskeletal tuberculosis. Despite antituberculous chemotherapy according to the current guidelines, the osteodestructive lesion progressed. This case highlights the difficulty of treating bone infections in general. Moreover, Mycobacterium tuberculosis as a rare causative agent of bone infections is discussed.