How to accelerate antimicrobial susceptibility testing.

BACKGROUND: Antimicrobial susceptibility testing (AST) results are crucial for timely administration of effective antimicrobial treatment, and, thus, should be made available to clinicians as fast as possible. In particular, increasing rates of multidrug-resistant organisms emphasize the need for rapid AST (rAST). OBJECTIVES: This article aims to provide microbiologists and clinicians with a critical overview of the current state of possibilities to accelerate AST. We also intend to discuss technical and strategic aspects of rAST, which may be helpful to academic researchers and assay developers in the industry. SOURCES: We have reviewed literature on rAST methods and their implementation in routine diagnostics. CONTENT: Phenotypic rAST is universal, mechanism-independent and allows exact categorization, but it demands time for the microorganisms to start the growth and to express the response to antibiotics. Detection of selected resistance mechanisms is more rapid, but the interpretation of its clinical impact is limited. Technical challenges of phenotypic rAST include inoculum effect, delayed expression of resistance, lag phase and initial biomass increase in susceptible isolates. Criteria for a successful rAST assay are ease of use, random access, capacity for simultaneous testing of multiple specimens, affordability and financial attractiveness for industry. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based AST seems to be particularly promising, as it can optimally be combined with MALDI-TOF MS identification. Direct testing from clinical specimens provides particularly early findings, with positive blood cultures being the most suitable specimen type. Polymicrobial samples and inoculum effect are serious obstacles for direct AST from other clinical specimens. Next to the technology improvement, optimization of pre-analytics and laboratory organization is essential. IMPLICATIONS: It appears feasible to generate an AST report within the same working shift; however, only affordable and easy-to-use rAST technologies have a chance to enter broad diagnostic routine. Efforts should be made by industry, authorities and academia to enable wide dissemination of rAST in clinical diagnostics.

Fungal prosthetic joint infection in total hip or knee arthroplasty: a retrospective single-centre study of 26 cases.

AIMS: Fungal prosthetic joint infections (PJIs) are rare and account for about 1% of total PJIs. Our aim was to present clinical and microbiological results in treating these patients with a two-stage approach and antifungal spacers. PATIENTS AND METHODS: We retrospectively reviewed our institutional database and identified 26 patients with positive fungal cultures and positive Musculoskeletal Infection Society (MSIS) criteria for PJI who were treated between 2009 and 2017. We identified 18 patients with total hip arthroplasty (THA) and eight patients with total knee arthroplasty (TKA). The surgical and antifungal treatment, clinical and demographic patient data, complications, relapses, and survival were recorded and analyzed. RESULTS: The median follow-up was 33 months. The success rate was 38.5% (10/26). Fluconazole resistance was found in 15%. Bacterial co-infection was common in 44% of patients for THA and 66% of patients with TKA. Mortality, reoperations, and treatment failure were common complications. CONCLUSION: Treatment with a two-stage exchange is a possible option for treatment, although fungal infections have a high failure rate. Therapeutic factors for treatment success remain unclear. Cite this article: Bone Joint J 2019;101-B:589-595.

Microbiological diagnostics of bloodstream infections in Europe-an ESGBIES survey.

OBJECTIVES: High-quality diagnosis of bloodstream infections (BSI) is important for successful patient management. As knowledge on current practices of microbiological BSI diagnostics is limited, this project aimed to assess its current state in European microbiological laboratories. METHODS: We performed an online questionnaire-based cross-sectional survey comprising 34 questions on practices of microbiological BSI diagnostics. The ESCMID Study Group for Bloodstream Infections, Endocarditis and Sepsis (ESGBIES) was the primary platform to engage national coordinators who recruited laboratories within their countries. RESULTS: Responses were received from 209 laboratories in 25 European countries. Although 32.5% (68/209) of laboratories only used the classical processing of positive blood cultures (BC), two-thirds applied rapid technologies. Of laboratories that provided data, 42.2% (78/185) were able to start incubating BC in automated BC incubators around-the-clock, and only 13% (25/192) had established a 24-h service to start immediate processing of positive BC. Only 4.7% (9/190) of laboratories validated and transmitted the results of identification and antimicrobial susceptibility testing (AST) of BC pathogens to clinicians 24 h/day. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry from briefly incubated sub-cultures on solid media was the most commonly used approach to rapid pathogen identification from positive BC, and direct disc diffusion was the most common rapid AST method from positive BC. CONCLUSIONS: Laboratories have started to implement novel technologies for rapid identification and AST for positive BC. However, progress is severely compromised by limited operating hours such that current practice of BC diagnostics in Europe complies only partly with the requirements for optimal BSI management.

Rapid detection of antibiotic resistance by MALDI-TOF mass spectrometry using a novel direct-on-target microdroplet growth assay.

OBJECTIVES: We aimed to develop a universal phenotypic method, which allows easy and rapid antimicrobial susceptibility testing independently of underlying resistance mechanisms. METHODS: We established a novel direct-on-target microdroplet growth assay for the detection of antibiotic resistance within a few hours, which is based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The microorganisms were incubated with and without meropenem in nutrient broth as microdroplets directly on MALDI-TOF MS target. Subsequently, broth was separated from microbial cells by contacting the microdroplets with an absorptive material. The microorganisms grown in the presence of antibiotic were detected by MALDI-TOF MS. A total of 24 Klebsiella pneumoniae and 24 Pseudomonas aeruginosa isolates were used to assess performance for detection of meropenem resistance. The microdroplet volumes investigated were 2, 4, 6, 8 and 10 µL. RESULTS: The best performance was achieved using 6-µL microdroplets. Applying this volume, all growth controls were successfully detected (definition of valid test), and all isolates were correctly categorized as susceptible or non-susceptible after an 18-h incubation. For K. pneumoniae, rate of valid tests, sensitivity and specificity all reached 100% after a 4-h incubation of 6-µL microdroplets. Using the same microdroplet volume for P. aeruginosa, incubation for 5 h resulted in 83.3% of valid tests with 100% sensitivity and 100% specificity. CONCLUSIONS: We demonstrated easy, rapid and accurate resistance detection using carbapenem-resistant Gram-negative bacteria as an example. Our technology is suitable for automatization and expandable to further applications, e.g. simultaneous testing of multiple antibiotics as well as resistance determination directly from clinical samples.

First description of an Anaerobiospirillum succiniciproducens prosthetic joint infection.

Anaerobiospirillum succiniciproducens belongs to the normal flora of cats and dogs and can rarely infect humans. Here, we report the first case of an A. succiniciproducens prosthetic joint infection.

Direct blood culturing on solid medium outperforms an automated continuously monitored broth-based blood culture system in terms of time to identification and susceptibility testing.

Pathogen identification and antimicrobial susceptibility testing (AST) should be available as soon as possible for patients with bloodstream infections. We investigated whether a lysis-centrifugation (LC) blood culture (BC) method, combined with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) identification and Vitek 2 AST, provides a time advantage in comparison with the currently used automated broth-based BC system. Seven bacterial reference strains were added each to 10 mL human blood in final concentrations of 100, 10 and 1 CFU/mL. Inoculated blood was added to the Isolator 10 tube and centrifuged at 3000 g for 30 min, then 1.5 mL sediment was distributed onto five 150-mm agar plates. Growth was observed hourly and microcolonies were subjected to MALDI-TOF MS and Vitek 2 as soon as possible. For comparison, seeded blood was introduced into an aerobic BC bottle and incubated in the BACTEC 9240 automated BC system. For all species/concentration combinations except one, successful identification and Vitek 2 inoculation were achieved even before growth detection by BACTEC. The fastest identification and inoculation for AST were achieved with Escherichia coli in concentrations of 100 CFU/mL and 10 CFU/mL (after 7 h each, while BACTEC flagged respective samples positive after 9.5 h and 10 h). Use of the LC-BC method allows skipping of incubation in automated BC systems and, used in combination with rapid diagnostics from microcolonies, provides a considerable advantage in time to result. This suggests that the usefulness of direct BC on solid medium should be re-evaluated in the era of rapid microbiology.

Successful interdisciplinary radical treatment of Mycobacterium fortuitum infection in a lipotourist from Germany after abdominoplasty in Turkey.

We report a case of a 30-year-old woman who experienced recurrent infections of the abdominal wall after travelling to Turkey from Germany to undergo abdominoplasty for aesthetic reasons. The patient's Mycobacterium fortuitum infection was successfully treated by surgery and antibiotic therapy. Surgical tourism-in this case, lipotourism-is resulting in an increasing number of patients in Europe who may present uncommon disease patterns.

Rapid identification of microorganisms from positive blood cultures by MALDI-TOF mass spectrometry subsequent to very short-term incubation on solid medium.

Rapid identification of the causative microorganism is important for appropriate antimicrobial therapy of bloodstream infections. Bacteria from positive blood culture (BC) bottles are not readily available for identification by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Lysis and centrifugation procedures suggested for direct MALDI-TOF MS from positive BCs without previous culture are associated with additional hands-on processing time and costs. Here, we describe an alternative approach applying MALDI-TOF MS from bacterial cultures incubated very briefly on solid medium. After plating of positive BC broth on Columbia blood agar (n = 165), MALDI-TOF MS was performed after 1.5, 2, 3, 4, 5, 6, 7, 8, 12 and (for control) 24 h of incubation until reliable identification to the species level was achieved (score ≥2.0). Mean incubation time needed to achieve species-level identification was 5.9 and 2.0 h for Gram-positive aerobic cocci (GPC, n = 86) and Gram-negative aerobic rods (GNR, n = 42), respectively. Short agar cultures with incubation times ≤2, ≤4, ≤6, ≤8 and ≤12 h yielded species identification in 1.2%, 18.6%, 64.0%, 96.5%, 98.8% of GPC, and in 76.2%, 95.2%, 97.6%, 97.6%, 97.6% of GNR, respectively. Control species identification at 24 h was achieved in 100% of GPC and 97.6% of GNR. Ethanol/formic acid protein extraction performed for an additional 34 GPC isolates cultivated from positive BCs showed further reduction in time to species identification (3.1 h). MALDI-TOF MS using biomass subsequent to very short-term incubation on solid medium allows very early and reliable bacterial identification from positive BCs without additional time and cost expenditure.

[Successful treatment of Fusarium-associated keratitis with multiresistant pathogen and multimorbid patient]. / Erfolgreiche Therapie einer Fusarium-assoziierten Keratitis bei multiresistentem Erreger und multimorbidem Patienten.

A 75-year-old man (not a contact lens wearer) presented with Fusarium-associated hypopyon keratitis. After several weeks of empirical and subsequently targeted antimycotic treatment, no considerable improvement was observed. However, after sclerokeratoplasty (11.2 × 11.2 mm) combined with prolonged antimycotic therapy a good local result with relapse-free long-term follow-up was achieved.

Trends in antimicrobial non-susceptibility in methicillin-resistant Staphylococcus aureus from Germany (2004-2011).

We analysed trends in antimicrobial non-susceptibility in methicillin-resistant Staphylococcus aureus (MSRA) from Germany to assess the impact of the changing population structure of MRSA on antimicrobial resistance rates. During two large nationwide multicentre studies in 2004-2005 and 2010-2011, we collected consecutively spa-genotyped MRSA isolates. The increase in non-susceptibility rates for tetracycline and trimethoprim-sulphamethoxazole was associated with the spread of livestock-associated MRSA. A decrease in non-susceptibility rates for aminoglycosides and quinolones affected all major lineages (spa-clonal complexes 003, 008, and 032). All isolated remained susceptible to glycopeptides and linezolid.