Rapid Discrimination of Gram-Positive and Gram-Negative Bacteria in Liquid Samples by Using NaOH-Sodium Dodecyl Sulfate Solution and Flow Cytometry

Abstract

BackgroundFor precise diagnosis of urinary tract infections (UTI), and selection of the appropriate prescriptions for their treatment, we explored a simple and rapid method of discriminating gram-positive and gram-negative bacteria in liquid samples.Methodology/Principal FindingsWe employed the NaOH-sodium dodecyl sulfate (SDS) solution conventionally used for plasmid extraction from Escherichia coli and the automated urine particle analyzer UF-1000i (Sysmex Corporation) for our novel method. The NaOH-SDS solution was used to determine differences in the cell wall structures between gram-positive and gram-negative bacteria, since the tolerance to such chemicals reflects the thickness and structural differences of bacterial cell walls. The UF-1000i instrument was used as a quantitative bacterial counter. We found that gram-negative bacteria, including E. coli, in liquid culture could easily be lysed by direct addition of equal volumes of NaOH-SDS solution. In contrast, Enterococcus faecalis, which is a gram-positive bacterium, could not be completely lysed by the solution. We then optimized the reaction time of the NaOH-SDS treatment at room temperature by using 3 gram-positive and 4 gram-negative bacterial strains and determined that the optimum reaction time was 5 min. Finally, in order to evaluate the generalizability of this method, we treated 8 gram-positive strains and 8 gram-negative strains, or 4 gram-positive and 4 gram-negative strains incubated in voluntary urine from healthy volunteers in the same way and demonstrated that all the gram-positive bacteria were discriminated quantitatively from gram negative bacteria using this method.Conclusions/SignificanceUsing our new method, we could easily discriminate gram-positive and gram-negative bacteria in liquid culture media within 10 min. This simple and rapid method may be useful for determining the treatment course of patients with UTIs, especially for those without a prior history of UTIs. The method may be easily applied in order to obtain additional information for clinical prescriptions from bacteriuria.

New and developing diagnostic technologies for urinary tract infections

“…Thus, these methods cannot be used for definitive diagnosis, but they might provide additional objective guidance for antibiotic prescription. Further attempts are being made to incorporate some speciation information into light scattering techniques, such as differential lysis of Gram-positive and Gram-negative bacteria with sodium dodecyl sulfate 53 and algorithms to distinguish the light-scattering patterns of bacilli from cocci 54 , but these techniques are still experimental.…”

Flow cytometric analysis of viable bacteria in urine samples of febrile patients at the emergency department

“…Using (viability) testing of bacteria the antibiotic resistance of the bacteria can be investigated (25,26). Also, Gram positive and negative bacteria can be distinguished by applying the technique of Wada (27) or Gessoni (28) in the FC analysis.…”

Flow Cytometry Analysis Using Sysmex UF-1000i Classifies Uropathogens Based on Bacterial, Leukocyte, and Erythrocyte Counts in Urine Specimens among Patients with Urinary Tract Infections

“…First, it has the ability to separate Gram-negative from Grampositive bacteria based on bacterial counts (31,32). Second, it can predict bacterial species (e.g., Citrobacter spp.)…”