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.
Highlights
During the initial treatment of infectious diseases, including urinary tract infections (UTIs), physicians prescribe antibiotics empirically because of a lack of information on the pathogen
We did not know whether the solution would lyse bacteria when it was directly added to the growth medium
In order to confirm if the NaOH-sodium dodecyl sulfate (SDS) lysis solution was able to lyse E. coli when it was directly added to the culture media and to confirm if the UF-1000i could measure the differences that were caused by the addition of the solution, we performed the following experiment
Summary
During the initial treatment of infectious diseases, including urinary tract infections (UTIs), physicians prescribe antibiotics empirically because of a lack of information on the pathogen. The gold standard for bacterial detection and enumeration is a semiquantitative urinary culture, the detection and enumeration requires an incubation that is at least overnight, and the identification of the relevant bacteria and susceptibility testing takes an additional 24 to 48 h For these reasons, information about the pathogen from laboratory cultures is usually not available at the first visit of the patient with a UTI. Urinary gram stain is a reliable diagnostic test for detection of bacteria in UTIs. Detection of bacteriuria by microscopy with Gram stain is the single best test and, if available locally and reported rapidly, is the only test that would need to be done to guide empirical treatment of patients with antibiotics [7]. For 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
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