Time to Positivity of Bacteria Cultures in Peritoneal Dialysis Fluid: Evaluation of Different Laboratory Techniques.

Patients with chronic kidney disease on peritoneal dialysis (PD) are susceptible to infections, with peritonitis being the primary cause of dropout. Peritoneal fluid culture is one of the essential elements for proper diagnosis and peritonitis treatment. The aim of this study was to compare the time required to obtain a positive culture using different laboratory methods. An in vitro cross-sectional study was conducted comparing different techniques for preparation and culture of bacteria in peritoneal fluid. The research was carried out with 21 sterile dialysis bags and 21 PD bags containing peritoneal fluid drained from patients without peritonitis. Fluids from the 42 PD bags were contaminated by injecting a coagulase-negative Staphylococcus suspension and then prepared for culture using 4 distinct techniques: A - direct culture; B - post-centrifugation culture; C - direct culture after 4 h sedimentation; and D - culture after 4 h sedimentation and centrifugation. This was followed by seeding. In the 21 contaminated sterile bags, mean times to obtain a positive culture with techniques D (19.6 h ± 2.6) and C (19.1 h ± 2.3) were longer than with technique A (15.8 h ± 3.0; p < 0.01), but not statistically different from group B (19.0 h ± 3.2). The same occurred in the 21 bags drained from patients, with mean times for techniques D (14.0 h ± 1.9) and C (14.5 h ± 1.7) being longer than technique A (12.22 h ± 1.94; p < 0.05) but not statistically different from technique B (13.2 h ± 1.3). The sedimentation and centrifugation steps seem to be unnecessary and may delay antibiotic sensitivity test results by approximately 8 hours.

Effects of meropenem exposure in persister cells of Acinetobacter calcoaceticus-baumannii.

AIM: To evaluate the influence of meropenem in the Acinetobacter calcoaceticus-baumannii (ACB) persister levels. METHODS: Persister levels in planktonic and biofilm cultures from ACB isolates were evaluated after exposure to different meropenem concentrations. RESULTS: A high variability of persister fractions was observed among the isolates cultured under planktonic and biofilm conditions. Meropenem concentration did not influence persister fractions, even when far above the MIC. No correlation was found between persister levels and biofilm biomass. CONCLUSION: The magnitude of persister levels from ACB planktonic and, particularly, biofilm cultures exposed to meropenem was independent of the antibiotic concentration, dosing regimen and biofilm biomass. These findings, in a context of meropenem failure to treat chronic infections, strengthen the importance of understanding persister behavior.

Real time PCR quantification of viable Mycobacterium tuberculosis from sputum samples treated with propidium monoazide.

Diagnostic methods of TB, nowadays, are prone to delay in diagnosis, increased false negative results and are not sensitive to many forms of paucibacillary disease. The aims of this study were to implement a quantitative nucleic acid-based diagnostic test for paucibacillary tuberculosis, enabling the identification and quantification of viable Mycobacterium tuberculosis bacilli by quantitative Real-Time PCR (qRT-PCR). The intergenic region of the single-copy inhA-mabA gene was chosen as the target region for design of primers and probes conjugated with fluorophores. The construction of synthetic DNA flanking the target region served as standards for absolute quantification of nucleic acids. Using the intercaling dye, propidium monoazide, we were able to discriminate between viable and dead cells of M. tuberculosis. The diagnosis method showed a broad sensitivity (96.1%) when only compared to samples of smear-positive sputum and ROC analyses shows that our approach performed well and yielded a specificity of 84.6% and a sensitivity of 84.6% when compared to M. tuberculosis colony-forming units counting.

Heterogeneous persister cells formation in Acinetobacter baumannii.

Bacterial persistence is a feature that allows susceptible bacteria to survive extreme concentrations of antibiotics and it has been verified in a number of species, such as Escherichia coli, Pseudomonas aeruginosa, Staphylococcus spp., Mycobacterium spp. However, even though Acinetobacter baumannii is an important nosocomial pathogen, data regarding its persistence phenotype are still lacking. Therefore, the aim of this study was to evaluate the persistence phenotype in A. baumannii strains, as well as its variation among strains after treatment with polymyxin B and tobramycin. Stationary cultures of 37 polymyxin B-susceptible clinical strains of A. baumannii were analyzed for surviving cells after exposure to 15 µg/mL of polymyxin B for 6 h, by serial dilutions and colony counting. Among these, the 30 tobramycin-susceptible isolates also underwent tobramycin treatment at a concentration of 160 µg/mL and persister cells occurrence was evaluated equally. A high heterogeneity of persister cells formation patterns among isolates was observed. Polymyxin B-treated cultures presented persister cells corresponding from 0.0007% to 10.1% of the initial population and two isolates failed to produce detectable persister cells under this condition. A high variability could also be observed when cells were treated with tobramycin: the persister fraction corresponded to 0.0003%-11.84% of the pre-treatment population. Moreover, no correlation was found between persister subpopulations comparing both antibiotics among isolates, indicating that different mechanisms underlie the internal control of this phenotype. This is the first report of persister cells occurrence in A. baumannii. Our data suggest that distinct factors regulate the tolerance for unrelated antibiotics in this species, contrasting the multi-drug tolerance observed in other species (eg. dormancy-mediated tolerance). Supporting this observation, polymyxin B--an antibiotic that is believed to act on non-dividing cells as well--failed to eradicate persister cells in the majority of the isolates, possibly reflecting a disconnection between persistence and dormancy.