Development of a rapid and simplified protocol for direct bacterial identification from positive blood cultures by using matrix assisted laser desorption ionization time-of- flight mass spectrometry

Background Bloodstream infections represent serious conditions carrying a high mortality and morbidity rate. Rapid identification of microorganisms and prompt institution of adequate antimicrobial therapy is of utmost importance for a successful outcome. Aiming at the development of a rapid, simplified and efficient protocol, we developed and compared two in-house preparatory methods for the direct identification of bacteria from positive blood culture flasks (BD BACTEC FX system) by using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF MS). Both methods employed saponin and distilled water for erythrocyte lysis. In method A the cellular pellet was overlaid with formic acid on the MALDI TOF target plate for protein extraction, whereas in method B the pellet was exposed to formic acid followed by acetonitrile prior to placing on the target plate. Results Best results were obtained by method A. Direct identification was achieved for 81.9 % and 65.8 % (50.3 % and 26.2 % with scores >2.0) of organisms by method A and method B, respectively. Overall concordance with final identification was 100 % to genus and 97.9 % to species level. By applying a lower cut-off score value, the levels of identification obtained by method A and method B increased to 89.3 % and 77.8 % of organisms (81.9 % and 65.8 % identified with scores >1.7), respectively. Using the lowered score criteria, concordance with final results was obtained for 99.3 % of genus and 96.6 % of species identifications. Conclusion The reliability of results, rapid performance (approximately 25 min) and applicability of in-house method A have contributed to implementation of this robust and cost-effective method in our laboratory

Sonication is superior to scraping for retrieval of bacteria in biofilm on titanium and steel surfaces in vitro

Background and purpose Low-virulence implant infections are characterized by bacterial colonization of the implant with subsequent biofilm formation. In these cases, soft tissue biopsies often prove to be culture negative. Consequently, detachment of the causative adherent bacteria is crucial for correct microbiological diagnosis. Using an in vitro model, we compared 4 methods of biofilm sampling from metal surfaces

Establishment of an in vivo rat model for chronic musculoskeletal implant infection

Background The aim of the study was to establish an experimental chronic musculoskeletal infection model in vivo characterized by (a) a small bacterial inoculum, (b) no general or local signs of infection, (c) several parallels (implants) in each animal and finally (d) a model that is technically easy to perform. Methods Bone xenografts with steel plates were implanted intramuscularly in rats. To the xenografts, different inocula of Staphylococcus aureus and two strains of Staphylococcus epidermidis were added. The animals were observed for different time periods before the removal of the xenografts. The xenografts and steel plates were subjected to quantitative bacterial culture after sonication. Additional steel plates were subjected to scanning electron microscopy (SEM) for visualization of biofilm formation. Results Inoculation of bone grafts with S. aureus did produce a pyogenic infection in all animals. A chronic infection was established in rats where the bone grafts were inoculated with S. epidermidis. A bacterial inoculum of 100 colony-forming units (CFU) of S. epidermidis was adequate as a minimum infective dose. During a period of up until 42 days, the animals infected with S. epidermidis had no general or local signs of infection. According to the results of the quantitative bacterial culture of sonicate fluid and SEM, a biofilm was developed on all implants. Conclusion In the present in vivo model, a very small bacterial inoculum succeeded in establishing a chronic musculoskeletal implant infection where a biofilm was formed on the implants. The experimental model is easy to perform and allows several implants in each animal. The model could be useful for the study of biofilm formation in vivo on different implants and different surfaces

Polymerase chain reaction versus culture in the diagnosis of Helicobacter pylori infection

In the management of <em>Helicobacter pylori</em>-induced gastroduodenal disease, a pilot study at our hospital (St. Olavs Hospital, Trondheim University Hospital, Trondheim, Norway) revealed that culture often seemed to fail compared to the polymerase chain reaction (PCR). A more thorough evaluation was therefore undertaken. We included 201 patients referred to upper gastrointestinal endoscopy in the period 2002-2004. Serology, biopsy rapid urease test, culture and PCR were performed. Conventional PCR was performed using the <em>ureC</em>, <em>vacA</em> and <em>cagA</em> genes, and real-time PCR for <em>ureC</em>. A diagnostic standard was defined on the basis of all four tests, and all four tests were then compared to this standard. One hundred eleven patients were deemed <em>H. pylori-</em>positive by the defined diagnostic standard, and 90 were labeled negative. Compared to this standard, culture showed a sensitivity of 87.4%, which was significantly lower than PCR at 99.1% (P&lt;0.001). Culture showed a perfect specificity of 100%, which was significantly better than PCR at 97.8%. <em>ureC</em> was the gene with the best sensitivity (94.6% in conventional PCR, 97.3% in real-time PCR). <em>vacA</em> sensitivity was 87.4%, which is significantly lower than ureC (P&lt;0.001). <em>cagA</em> was present in 37.8% of our <em>H. pylori</em>-positive patients. By real-time PCR a significantly lower cycle threshold was observed in antral biopsies than in corpal biopsies, indicating a higher <em>H. pylori</em> DNA template concentration in antral biopsies. PCR-testing for <em>H. pylori</em> is faster and significantly more sensitive than culture. Culture on the other hand was significantly more specific than PCR in our hand

Peritoneal Dialysis-Associated Peritonitis Caused by Dermabacter hominis

Dermabacter hominis was the cause of a peritoneal dialysis-associated peritonitis. D. hominis was identified by phenotypic criteria and by sequencing the 16S rRNA gene. Clinical cure was achieved with cefuroxime treatment despite the isolate's reduced susceptibility to this drug (MIC, 12 mg/liter) on in vitro testing. The successful treatment was probably due to the high concentrations attained by intraperitoneal administration of the drug

Rapid Multiple-Locus Variant-Repeat Assay (MLVA) for Genotyping of Streptococcus agalactiae▿

Several methods have been used for typing of Streptococcus agalactiae (group B streptococci [GBS]). Methods currently in use may provide inadequate resolution (e.g., typing of capsular polysaccharides and surface protein) or are labor-intensive and expensive (e.g., multilocus sequence typing [MLST] or pulsed-field gel electrophoresis). This work describes the construction and use of a multiple-locus variant-repeat assay (MLVA) on 126 well-characterized human GBS strains, consisting mostly of invasive Norwegian strains and international reference strains. Based on in silico whole-genomic analysis of the genomes of strains A909, NEM316, and 2603V/R, 18 candidate loci were selected and investigated by PCR. Eleven loci showed diversity, and the five most diverse loci were used for the construction of an MLVA, consisting of a multiplex PCR followed by fragment analysis with capillary electrophoresis. The assay generated clusters which corresponded well with those observed by other methods. However, it provided a considerably higher degree of diversity, with 70 different MLVA types compared to 36 types generated by MLST. Simpson's index of diversity for the 5-locus MLVA was 0.963, compared to 0.899 for the MLST in this strain collection. MLVA results will generally be available within 2 days, which is usually faster than MLST. In our hands, MLVA of GBS represents a rapid, easy, and comparably inexpensive method for high-resolution genotyping of GBS

Characterization of the virulence potential of Staphylococcus condimenti isolated from a patient with severe soft tissue infection

The coagulase-negative bacterium Staphylococcus condimenti and closely related species are commonly isolated from or found in starter cultures of fermented sausage as well as fish and soy sauces, and have traditionally been considered nonpathogenic. Recently, however, a case of catheter-related bacteraemia caused by S. condimenti was reported. In the present study we identified and characterized a strain of S. condimenti isolated from a patient with a severe soft tissue infection, comparing it to S. condimenti and S. carnosus type strains in order to elucidate the virulence potential of the clinical strain. Genome comparison showed high degree of conservation between the clinical strain and the type strain used in food industry, as well as with S. carnosus. The genome of the clinical S. condimenti strain contains few horizontally transferred regions and 37 putative virulence genes, including genes with similarity to leucocidin and genes involved in immune evasion, proinflammatory and cytolytic activity. However, it remains to be tested whether these putative virulence genes are expressed and functional. Although uncommon, S. condimenti may cause severe infection in previously healthy persons