Rapid prototyping vaccine approach in mice against multi-drug resistant Gram-negative organisms from clinical isolates based on outer membrane vesicles.

Hospital-acquired infections due to multi-drug resistant Gram-negative organisms (MDRGNO) pose a major threat to global health. A vaccine preventing colonization and consecutive infection with MDRGNO could be particularly valuable, as therapeutic options become increasingly limited. Outer membrane vesicles (OMV) of Escherichia coli strain CFT073 as well as three MDRGNO strains that had caused severe infections in humans were administered intranasally to mice, with and without cholera toxin as an adjuvant. The humoral immune responses were comparatively matched with the sera of patients, who had suffered an infection caused by the respective bacterium. Additionally, systemic and local toxicity was evaluated. Intranasal vaccination with OMV could elicit solid humoral immune responses (total IgM and IgG), specific for the respective MDRGNO in mice; decoration of vital bacterial membranes with antibodies was comparable to patients who had survived systemic infection with the respective bacterial isolate. After intranasal vaccination of mice with OMV no signs of local or systemic toxicity were observed. Intranasal vaccination with OMV may open up a rapid vaccine approach to prevent colonization and/or infection with pathogenic MDRGNOs, especially in an outbreak setting within a hospital. It may also be an option for patients who have to undergo elective interventions in centers with a high risk of infection for certain common MDRGNO. Future studies need to include challenge experiments as well as phase I trials in humans.

First report on bla NDM-1-producing Acinetobacter baumannii in three clinical isolates from Ethiopia.

BACKGROUND: Multidrug-resistant Gram-negative bacterial infections are recognized as one of the major threats to global health. In this study, we describe for the first time bla NDM-1 gene carrying organisms from Ethiopia consisting of three Acinetobacter baumannii isolates from patients in Jimma. METHODS: Besides phenotypic antimicrobial susceptibility testing, molecular strain typing and sequencing was performed to describe the phylogenetic relation of the Ethiopian isolates in detail in relation to published isolates from all over the globe. RESULTS AND DISCUSSION: Three multi-resistant, bla NDM-1-positive Acinetobacter baumannii isolates, most likely a local clonal diffusion, were isolated. Two of the three isolates described within this study were untreatable with the locally available antimicrobials and were only susceptible to polymyxin B and amikacin. The genome sequences confirmed the isolates to be distinct from the outbreak strains reported from Kenya, the only other characterized bla NDM-1 positive Acinetobacter baumannii strains in East Africa so far. Up to date, no other bacterial species were found to harbour the gene cassette in Jimma and conjugation to E. coli was not successful under laboratory conditions. However, natural transmission to other bacteria seems likely, given the evident lack of hygienic precautions due to limited resource settings. CONCLUSIONS: The detected isolates could solely be the tip of the iceberg regarding the presence of NDM-1 producing organisms in the region, as only a limited number of bacterial isolates were evaluated so far and until recently, susceptibility testing and isolation of bacteria could hardly be performed in clinical patient care. These multi-drug resistant organisms pose a serious threat to antimicrobial treatments in Jimma, Ethiopia.

Evaluation of matrix-assisted laser desorption ionization-time of flight mass spectrometry for rapid detection of ß-lactam resistance in Enterobacteriaceae derived from blood cultures.

The identification of pathogens directly from blood cultures by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) can be a valuable tool for improving the treatment of patients with sepsis and bacteremia. However, the increasing incidence of multidrug-resistant Gram-negative bacteria makes it difficult to predict resistance patterns based only on pathogen identification. Most therapy regimens for sepsis caused by Gram-negative rods consist of at least one ß-lactam antibiotic. Thus, it would be of great benefit to have an early marker of resistance against these drugs. In the current study, we tested 100 consecutive blood cultures containing Enterobacteriaceae for resistance against 3rd-generation cephalosporins in a MALDI-TOF MS ß-lactamase assay. Escherichia coli was also tested for resistance against aminopenicillins. The results of the ß-lactamase assay were compared with those of conventional methods. The assay permitted discrimination between E. coli strains that were resistant or susceptible to aminopenicillins with a sensitivity and a specificity of 100%. The same was true for resistance to 3rd-generation cephalosporins in Enterobacteriaceae that constitutively produced class C ß-lactamases. Discrimination was more difficult in species expressing class A ß-lactamases, as these enzymes can generate false-positive results. Thus, the sensitivity and specificity for this group were 100% and 91.5%, respectively. The test permitted the prediction of resistance within 2.5 h after the blood culture was flagged as positive.

Stability of gametocyte-specific Pfs25-mRNA in dried blood spots on filter paper subjected to different storage conditions.

BACKGROUND: Real-time quantitative nucleic acid sequence-based amplification (QT-NASBA) is a sensitive method for detection of sub-microscopic gametocytaemia by measuring gametocyte-specific mRNA. Performing analysis on fresh whole blood samples is often not feasible in remote and resource-poor areas. Convenient methods for sample storage and transport are urgently needed. METHODS: Real-time QT-NASBA was performed on whole blood spiked with a dilution series of purified in-vitro cultivated gametocytes. The blood was either freshly processed or spotted on filter papers. Gametocyte detection sensitivity for QT-NASBA was determined and controlled by microscopy. Dried blood spot (DBS) samples were subjected to five different storage conditions and the loss of sensitivity over time was investigated. A formula to approximate the loss of Pfs25-mRNA due to different storage conditions and time was developed. RESULTS: Pfs25-mRNA was measured in time to positivity (TTP) and correlated well with the microscopic counts and the theoretical concentrations of the dilution series. TTP results constantly indicated higher amounts of RNA in filter paper samples extracted after 24 hours than in immediately extracted fresh blood. Among investigated storage conditions freezing at -20°C performed best with 98.7% of the Pfs25-mRNA still detectable at day 28 compared to fresh blood samples. After 92 days, the RNA detection rate was only slightly decreased to 92.9%. Samples stored at 37°C showed most decay with only 64.5% of Pfs25-mRNA detectable after one month. The calculated theoretical detection limit for 24 h-old DBS filter paper samples was 0.0095 (95% CI: 0.0025 to 0.0380) per µl. CONCLUSIONS: The results suggest that the application of DBS filter papers for quantification of Plasmodium falciparum gametocytes with real-time QT-NASBA is practical and recommendable. This method proved sensitive enough for detection of sub-microscopic densities even after prolonged storage. Decay rates can be predicted for different storage conditions as well as durations.