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Staphylococcus aureus has acquired resistance to antibiotics since their first use. The S. aureus protein NorA, an efflux pump belonging to the major facilitator superfamily (MFS), contributes to resistance to fluoroquinolones (e.g., ciprofloxacin), biocides, dyes, quaternary ammonium compounds, and antiseptics. Different compounds have been identified as potential efflux pump inhibitors (EPIs) of NorA that result in increased intracellular concentration of antibiotics, restoring their antibacterial activity and cell susceptibility. However, none of the currently known EPIs have been approved for clinical use, probably due to their toxicity profiles. In the present study, we screened approved drugs for possible efflux pump inhibition. By screening a compound library of approximately 1200 different drugs, we identified nilotinib, a tyrosine kinase inhibitor, as showing the best efflux pump inhibitory activity, with a fractional inhibitory concentration index of 0.1875, indicating synergism with ciprofloxacin, and a minimum effective concentration as low as 0.195 μM. Moreover, at 0.39 μM, nilotinib, in combination with 8 μg/mL of ciprofloxacin, led to a significant reduction in biofilm formation and preformed mature biofilms. This is the first description of an approved drug that can be used as an efflux pump inhibitor and to reduce biofilms formation at clinically achievable concentrations.
Introduction: The environmental bacterium Burkholderia pseudomallei causes the often fatal and massively underreported infectious disease melioidosis. Antigens inducing protective immunity in experimental models have recently been identified and serodiagnostic tools have been improved. However, further elucidation of the antigenic repertoire of B. pseudomallei during human infection for diagnostic and vaccine purposes is required. The adaptation of B. pseudomallei to very different habitats is reflected by a huge genome and a selective transcriptional response to a variety of conditions. We, therefore, hypothesized that exposure of B. pseudomallei to culture conditions mimicking habitats encountered in the human host might unravel novel antigens that are recognized by melioidosis patients.
Methods and results: In this study, B. pseudomallei was exposed to various stress and growth conditions, including anaerobiosis, acid stress, oxidative stress, iron starvation and osmotic stress. Immunogenic proteins were identified by probing two-dimensional Western blots of B. pseudomallei intracellular and extracellular protein extracts with sera from melioidosis patients and controls and subsequent MALDI-TOF MS. Among B. pseudomallei specific immunogenic signals, 90 % (55/61) of extracellular immunogenic proteins were identified by acid, osmotic or oxidative stress. A total of 84 % (44/52) of intracellular antigens originated from the stationary growth phase, acidic, oxidative and anaerobic conditions. The majority of the extracellular and intracellular protein antigens were identified in only one of the various stress conditions. Sixty-three immunoreactive proteins and an additional 38 candidates from a literature screening were heterologously expressed and subjected to dot blot analysis using melioidosis sera and controls. Our experiments confirmed melioidosis-specific signals in 58 of our immunoproteome candidates. These include 15 antigens with average signal ratios (melioidosis:controls) greater than 10 and another 26 with average ratios greater than 5, including new promising serodiagnostic candidates with a very high signal-to-noise ratio.
Conclusion: Our study shows that a comprehensive B. pseudomallei immunoproteomics approach, using conditions which are likely to be encountered during infection, can identify novel antibody targets previously unrecognized in human melioidosis.
Analysis of volatile organic compounds (VOCs) is a novel approach to accelerate bacterial culture diagnostics of Mycobacterium avium subsp. paratuberculosis (MAP). In the present study, cultures of fecal and tissue samples from MAP-infected and non-suspect dairy cattle and goats were explored to elucidate the effects of sample matrix and of animal species on VOC emissions during bacterial cultivation and to identify early markers for bacterial growth. The samples were processed following standard laboratory procedures, culture tubes were incubated for different time periods. Headspace volume of the tubes was sampled by needle trap-micro-extraction, and analyzed by gas chromatography-mass spectrometry. Analysis of MAP-specific VOC emissions considered potential characteristic VOC patterns. To address variation of the patterns, a flexible and robust machine learning workflow was set up, based on random forest classifiers, and comprising three steps: variable selection, parameter optimization, and classification. Only a few substances originated either from a certain matrix or could be assigned to one animal species. These additional emissions were not considered informative by the variable selection procedure. Classification accuracy of MAP-positive and negative cultures of bovine feces was 0.98 and of caprine feces 0.88, respectively. Six compounds indicating MAP presence were selected in all four settings (cattle vs. goat, feces vs. tissue): 2-Methyl-1-propanol, 2-methyl-1-butanol, 3-methyl-1-butanol, heptanal, isoprene, and 2-heptanone. Classification accuracies for MAP growth-scores ranged from 0.82 for goat tissue to 0.89 for cattle feces. Misclassification occurred predominantly between related scores. Seventeen compounds indicating MAP growth were selected in all four settings, including the 6 compounds indicating MAP presence. The concentration levels of 2,3,5-trimethylfuran, 2-pentylfuran, 1-propanol, and 1-hexanol were indicative for MAP cultures before visible growth was apparent. Thus, very accurate classification of the VOC samples was achieved and the potential of VOC analysis to detect bacterial growth before colonies become visible was confirmed. These results indicate that diagnosis of paratuberculosis can be optimized by monitoring VOC emissions of bacterial cultures. Further validation studies are needed to increase the robustness of indicative VOC patterns for early MAP growth as a pre-requisite for the development of VOC-based diagnostic analysis systems.
Melioidosis is a seasonal infectious disease in tropical and subtropical areas caused by the soil bacterium Burkholderia pseudomallei. In many parts of the world, including South West India, most cases of human infections are reported during times of heavy rainfall, but the underlying causes of this phenomenon are not fully understood. India is among the countries with the highest predicted melioidosis burden globally, but there is very little information on the environmental distribution of B. pseudomallei and its determining factors. The present study aimed (i) to investigate the prevalence of B. pseudomallei in soil in South West India, (ii) determine geochemical factors associated with B. pseudomallei presence and (iii) look for potential seasonal patterns of B. pseudomallei soil abundance. Environmental samplings were performed in two regions during the monsoon and post-monsoon season and summer from July 2016 to November 2018. We applied direct quantitative real time PCR (qPCR) together with culture protocols to overcome the insufficient sensitivity of solely culture-based B. pseudomallei detection from soil. A total of 1,704 soil samples from 20 different agricultural sites were screened for the presence of B. pseudomallei. Direct qPCR detected B. pseudomallei in all 20 sites and in 30.2% (517/1,704) of all soil samples, whereas only two samples from two sites were culture-positive. B. pseudomallei DNA-positive samples were negatively associated with the concentration of iron, manganese and nitrogen in a binomial logistic regression model. The highest number of B. pseudomallei-positive samples (42.6%, p < 0.0001) and the highest B. pseudomallei loads in positive samples [median 4.45 × 103 genome equivalents (GE)/g, p < 0.0001] were observed during the monsoon season and eventually declined to 18.9% and a median of 1.47 × 103 GE/g in summer. In conclusion, our study from South West India shows a wide environmental distribution of B. pseudomallei, but also considerable differences in the abundance between sites and within single sites. Our results support the hypothesis that nutrient-depleted habitats promote the presence of B. pseudomallei. Most importantly, the highest B. pseudomallei abundance in soil is seen during the rainy season, when melioidosis cases occur.
BK polyomavirus-associated haemorrhagic cystitis (BKHC) is a complication after allogeneic stem cell transplantation, which can occur in 5–60% of the cases. BK viruria alone can also occur in up to 100%. BKHC can lead to severe morbidity in stem cell-transplanted patients, but data about this disease is limited. Consequently, we conducted a prospective unicentric non-interventional trial on BKHC as well as BK viruria after first adult allogeneic stem cell transplantation with a follow-up time of 1 year after inpatient treatment. Between November 2013 and December 2015, we were able to include 40 adult patients with a mean age of 52.8 years. Twenty-seven (67.5%) of these patients were male and 13 (32.5%) were female. Acute myeloid leukaemia was the most frequent underlying disease (n = 15; 37.5%). Only 1 patient developed BKHC during inpatient treatment (n = 1; 2.5%), but BK viruria was frequent (n = 11; 27.5%) during inpatient treatment as well as in the follow-up time (n = 14; 35%). Interestingly, BK viruria was significantly associated with mucositis (p = 0.038) and number of transfused platelet concentrates (p = 0.001). This unexpected association will be discussed and needs further investigation.
Matrix-assisted laser desorption/ionization time-of-flight-mass spectrometry (MALDI-TOF MS)-based direct-on-target microdroplet growth assay (DOT-MGA) was recently described as a novel method of phenotypic antimicrobial susceptibility testing (AST). Here, we developed the application of MALDI-TOF MS-based DOT-MGA for Gram-positive bacteria including AST from agar cultures and directly from positive blood cultures (BCs) using the detection of methicillin resistance as example. Consecutively collected, a total of 14 methicillin-resistant Staphylococcus aureus (MRSA) and 14 methicillin-susceptible S. aureus (MSSA) clinical isolates were included. Furthermore, a collection of MRSA challenge strains comprising different SCCmec types, mec genes, and spa types was tested. Blood samples were spiked with MRSA and MSSA and positive BC broth processed by three different methods: serial dilution of BC broth, lysis/centrifugation, and differential centrifugation. Processed BC broth was directly used for rapid AST using DOT-MGA. Droplets of 6 μl with and without cefoxitin at the EUCAST breakpoint concentration were spotted in triplicates onto the surface of a MALDI target. Targets were incubated in a humidity chamber, followed by medium removal and on-target protein extraction with formic acid before adding matrix with an internal standard as a quality control (QC). Spectra were acquired and evaluated using MALDI Biotyper software. First, tests were considered as valid, if the growth control achieved an identification score of ≥1.7. For valid tests, same score criterion was used for resistant isolates when incubated with cefoxitin. An identification score <1.7 after incubation with cefoxitin defined susceptible isolates. On-target protein extraction using formic acid considerably improved detection of methicillin resistance in S. aureus and DOT-MGA showed feasible results for AST from agar cultures after 4 h incubation time. Comparing the different processing methods of positive BC broth, lysis/centrifugation method with a final dilution step 10–1 of the 0.5 McFarland suspension resulted in best test performance after 4 h incubation time. Overall, 96.4% test validity, 100% sensitivity, and 100% specificity were achieved for detection of methicillin resistance in clinical isolates. All strains of the MRSA challenge collection were successfully tested as methicillin-resistant. This first study on Gram-positive organisms showed feasibility and accuracy of MALDI-TOF MS-based DOT-MGA for rapid AST of S. aureus from agar cultures and directly from positive BCs.