Counting bacteria in microfluidic devices: Smartphone compatible 'dip-and-test' viable cell quantitation using resazurin amplified detection in microliter capillary arrays.

Viable bacterial cell counting is fundamental to analytical microbiology and agar plate colony counting remains common yet laborious and slow. Here, we demonstrate two methods for counting bacteria using commercially available microfluidic devices. We show that accurate viable cell counting is possible using simple and easy 'dip and test' arrays of microcapillaries. Colorimetric and fluorescent growth detection both permit viable cell counting in microcapillaries either by limiting dilution into multiple microfluidic compartments using a single endpoint measurement, or alternatively by quantifying growth kinetics. The microcapillary devices are compatible with conventional 96 well plates and multichannel pipettes, expanding each microplate row into 120 individual 1 or 2 µL samples. At limiting dilution, counting the proportion of positive compartments permitted accurate calculation of gram-negative and gram-positive bacteria (E. coli and S. saprophyticus) at concentrations down to as low as 10 CFU/mL with almost 1:1 agreement with agar plate colony counts over four orders of magnitude. A smartphone camera was sufficient to record endpoint images of resazurin growth detection both colorimetrically and fluorescently. Viable cell counting of E. coli and S. saprophyticus was also possible through recording growth kinetics and determining the time taken to detect resazurin conversion. However, only the limiting dilution method remained consistent in the presence of urine matrix, as some interference in growth rate was observed when bacteria were spiked into higher concentrations of normal urine to simulate urinary tract infection patient samples. However, with the limiting dilution counting method endpoint growth was always detected even in the presence of 90% urine matrix, suggesting that this method might permit bacterial pathogen counting directly in clinical samples without agar plating.

Biocompatible indocyanine green loaded PLA nanofibers for in situ antimicrobial photodynamic therapy.

Chronic wounds and their associated bacterial infections are major issues in modern health care systems. Therefore, antimicrobial resistance (AMR), treatment costs, and number of disability-adjusted life-years have gained more interest. Recently, photodynamic therapy emerged as an effective approach against resistant and naïve bacterial strains with a low probability of creating AMR. In this study, needleless electrospinning was used to produce an indocyanine green (ICG) loaded poly(d,l-lactide) nanofibrous mesh as a photoresponsive wound dressing. The non-woven mesh had a homogeneous nanofibrous structure and showed long-term hydrolytic stability at different pH values. The antimicrobial activity was tested against several bacterial strains, namely Staphylococcus saprophyticus subsp. bovis, Escherichia coli DH5 alpha, and Staphylococcus aureus subsp. aureus. Upon irradiation with a laser of a specific wavelength (λ = 810 nm), the bacterial viability was significantly reduced by 99.978% (3.66 log10), 99.699% (2.52 log10), and 99.977% (3.64 log10), respectively. The nanofibrous mesh showed good biocompatibility, which was confirmed by the proliferation of mouse fibroblasts (L929) on the surface and into deeper parts of the mesh. Furthermore, a favorable proangiogenic effect was observed in ovo using the chorioallantoic membrane assay. In general, it can be concluded that ICG loaded nanofibers as an innovative wound dressing represent a promising strategy against chronic wounds associated with skin infections.

Multimeric TAT peptides are effective in vitro inhibitors of Staphylococcus saprophyticus.

TAT (48-60) is a tridecapeptide from the envelope protein of HIV that was previously shown to possess cell-penetrating properties and antibacterial activity, making it a potential drug delivery agent for anticancer drugs and as antibacterial compound. Previous reports indicated that dimerization enhances the desired bioactivity of TAT; hence, we sought to synthesize multimeric TAT peptides. Herein, we describe the effects of multimerization on the antibacterial activity and secondary structure of the peptide. Terminal modifications such as N-acetylation and C-amidation were employed in the design. TATp monomer, dimer, and tetramer were synthesized using solid-phase peptide synthesis, purified by reversed-phase HPLC, and then characterized by mass spectrometry. Multimerization of the peptide did not change the secondary structure conformation. The CD analysis revealed a polyproline-II conformation for all peptide designs. Thus, this study provides a method of increasing the biological activity of the peptide by multimerization while retaining the secondary conformation of its monomeric unit. Furthermore, the bacteria Staphylococcus saprophyticus was found to be susceptible to the dimer and tetramer, with MIC50 of 12.50 µm and <1.56 µm, respectively. This suggests a structure-activity relationship whereby the antibacterial activity increases with increase in valency.

Inhibition of urease activity in the urinary tract pathogens Staphylococcus saprophyticus and Proteus mirabilis by dimethylsulfoxide (DMSO).

AIMS: Urease is a virulence factor for the urinary tract pathogens Staphylococcus saprophyticus and Proteus mirabilis. Dimethylsulfoxide (DMSO) is structurally similar to urea, used as a solvent for urease inhibitors, and an effective treatment for interstitial cystitis/bladder pain syndrome (IC/BPS). The aims of this study were to test DMSO as a urease inhibitor and determine its physiological effects on S. saprophyticus and P. mirabilis. METHODS AND RESULTS: Urease activity in extracts and whole cells was measured by the formation of ammonium ions. Urease was highly sensitive to noncompetitive inhibition by DMSO (Ki about 6 mmol l-1 ). DMSO inhibited urease activity in whole cells, limited bacterial growth in media containing urea, and slowed the increase in pH which occurred in artificial urine medium. CONCLUSIONS: DMSO should be used with caution as a solvent when testing plant extracts or other potential urease inhibitors. Because it can inhibit bacterial growth and delay an increase in pH, it may be an effective treatment for urinary tract infections. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first detailed study of the inhibition of urease by DMSO. Dimethylsulfoxide may be used to treat urinary tract infections that are resistant to antibiotics or herbal remedies.

The influence of pH on Staphylococcus saprophyticus iron metabolism and the production of siderophores.

Staphylococcus saprophyticus is a gram-positive coagulase negative bacteria which shows clinical importance due to its capability of causing urinary tract infections (UTI), as well as its ability to persist in this environment. Little is known about how S. saprophyticus adapts to the pH shift that occurs during infection. Thus, in this study we aim to use a proteomic approach to analyze the metabolic adaptations which occur as a response by S. saprophyticus when exposed to acid (5.5) and alkaline (9.0) pH environments. Proteins related to iron storage are overexpressed in acid pH, whilst iron acquisition proteins are overexpressed in alkaline pH. It likely occurs because iron is soluble at acid pH and insoluble at alkaline pH. To evaluate if S. saprophyticus synthesizes siderophores, CAS assays were performed, and the results confirmed their production. The chemical characterization of siderophores demonstrates that S. saprophyticus produces carboxylates derived from citrate. Of special note is the fact that citrate synthase (CS) is down-regulated during incubation at acid pH, corroborating this result. This data was also confirmed by enzymatic assay. Our results demonstrate that iron metabolism regulation is influenced by different pH levels, and show, for the first time, the production of siderophores by S. saprophyticus. Enzymatic assays suggest that citrate from the tricarboxylic acid cycle (TCA) is used as substrate for siderophore production.

Structural characterization and biological evaluation of Staphylosan (dimannooleate), a new glycolipid surfactant produced by a marine Staphylococcus saprophyticus SBPS-15.

Marine microbes have gained significant attention as potential biofactories for broad spectrum bioactive compounds. In the recent years, bioactive biosurfactants have warranted renewed interest from both environmental and clinical sectors as anti-biofilm agents due to their excellent properties of dispersing microbial biofilms. The present study explores a new glycolipid biosurfactant produced by a marine Staphylococcus saprophyticus exhibiting interesting biological activities. This glycolipid biosurfactant was purified and identified as Mannose-Mannose-Oleic acid (named as Staphylosan) based on the results of NMR, GC, GC-MS, MALDI-TOF-MS and tandem MS analysis. The surface tension and critical micelle concentration of purified Staphylosan was 30.9 mN m-1 and 24 mg L-1. Further, it showed promising biofilm inhibition and dislodging activities against a panel of profuse biofilm forming bacteria at both single and multi-species level which were isolated from boat hull biofilm environment such as Bacillus subtilis BHKH-7, Acinetobacter beijerinckii BHKH-11, Pseudomonas aeruginosa BHKH-19, Serratia liquefaciens BHKH-23, Marinobacter lipolyticus BHKH-31 and Micrococcus luteus BHKH-39. Moreover, it exhibited anionic charge and revealed non-toxicity towards brine shrimps, suggesting its environmental safety. This is a first report on Staphylosan, a multifunctional glycolipid surfactant from a marine Staphylococcus saprophyticus SBPS-15, exhibiting promising anti-biofilm activities and non-toxic in nature and thus finds possible potential use in many environmental applications especially under marine conditions.

The exoproteome profiles of three Staphylococcus saprophyticus strains reveal diversity in protein secretion contents.

Staphylococcus saprophyticus is a gram-positive microorganism responsible for urinary tract infections (UTIs). Although some virulence factors are characterized, such as urease, autolysins, adhesins and hemagglutinins, large-scale proteomic studies have not been performed within this species. We performed the characterization of the exoproteome from three S. saprophyticus strains: the reference strain ATCC 15,305, a non-capsular strain 7108 and the 9325 strain containing a thick capsule which were cultured in BHI medium and culture supernatants were analysed by using mass spectrometry approach. We observed a core of 72 secreted proteins. In addition, it was possible to detect diversity in the protein profiles of the exoproteomes. Interestingly, strain 7108 presented no secretion of three antigenic proteins, including the classical SsaA antigen. In addition, the level of antigenic proteins secreted by strain 9325 was higher than in ATCC 15,305. This result was confirmed by Western blot analysis using anti-SsaA polyclonal antibodies, and no production/ secretion of SsaA was detected in strain 7108. Transcriptional data shows that 7108 strain produces transcripts encoding SsaA, suggesting post-transcriptional regulation occurs in this strain. Moreover, when compared with the other strains that were analyzed, it was possible to detect higher levels of proteases secreted by strain 7108 and higher levels of antigenic proteins and transglycosylases secreted by 9325 strain. The results reveal diversity in protein secretion among strains. This research is an important first step towards understanding the variability in S. saprophyticus exoproteome profile and could be significant in explaining differences among strains.

Synthesis of the erythromycin-conjugated nanodendrimer and its antibacterial activity.

The development and spread of bacterial resistance to antimicrobial drugs necessitates the need to search for novel and effective antimicrobial agents. In the last few decades, innovative nanomaterials are attracting increasing attention and, among them, dendrimers have shown wide application in the various fields. In the current study, the two generations of an anionic linear- spherical nanodendrimer G1 and G2 were synthetized and compound G2 of nanodendrimer conjugated with erythromycin. The structures of the nanodendrimers were characterized by FTIR spectroscopy, zetasizer, and scanning electron microscopy (SEM). The antibacterial activity of the erythromycin-conjugated nanodendrimer and erythromycin alone were evaluated by the microdilution method against Staphylococcus aureus, S. epidermidis, S. saprophyticus, and Pseudomonas aeruginosa. The size of first and second generation of nanodendrimer, and the erythromycin-conjugated nanodendrimer was 75, 95, and 65.6 nm, respectively. The drug loading percentage of the nanodendrimer conjugates was obtained to be in 35.2%. In our study, the erythromycin-conjugated nanodendrimer showed significantly more bacteriostatic and bactericidal activities against all four studied bacteria than erythromycin alone. Our study's results highlight that the erythromycin-conjugated nanodendrimer is a highly effective agent against Gram positive and negative bacteria. The antibacterial properties of erythromycin combined with the targeting potential of the nanodendrimer can lead to sustained intracellular delivery of therapeutic agent.

Activities of Oxadiazole Antibacterials against Staphylococcus aureus and Other Gram-Positive Bacteria.

The activities of four oxadiazoles were investigated with 210 methicillin-resistant Staphylococcus aureus (MRSA) strains. MIC50 and MIC90 values of 1 to 2 and 4 µg/ml, respectively, were observed. We also evaluated the activity of oxadiazole ND-421 against other staphylococci and enterococci and in the presence of oxacillin for selected MRSA strains. The MIC for ND-421 is lowered severalfold in combination with oxacillin, as they synergize. The MIC90 of ND-421 against vancomycin-resistant enterococci is ≤1 µg/ml.

Evaluation of Lactic Acid Bacteria Isolated from Fermented Plant Products for Antagonistic Activity Against Urinary Tract Pathogen Staphylococcus saprophyticus.

Urinary tract infections (UTIs) are the most common infectious diseases in infants and the elderly; they are also the most common among nosocomial infections. The treatment of UTIs usually involves a short-term course of antibiotics. The purpose of this study was to identify the strains of lactic acid bacteria (LAB) that can inhibit the urinary tract pathogen Staphylococcus saprophyticus, as alternatives to antibiotics. In this study, we collected 370 LAB strains from fermented plant products and reference strains from the Bioresources Collection and Research Center (BCRC). Using spent culture supernatants (SCS), we then screened these LAB strains with for antimicrobial effects on urinary tract pathogens by the well-diffusion assay. Seven LAB strains-PM2, PM68, PM78, PM201, PM206, PM229, and RY2-exhibited inhibitory activity and were evaluated for anti-growth activity against urinary tract pathogens by the co-culture inhibition assay. Anti-adhesion and anti-invasion activities against urinary tract pathogens were evaluated using the SV-HUC-1 urothelial cell cultures. The results revealed that the survival rate of S. saprophyticus ranged from 0.9-2.96%, with the pH continuously decreasing after co-culture with LAB strains for 4 h. In the competitive adhesion assay, the exclusion and competition groups performed better than the displacement group. In the SV-HUC-1 cell invasion assay, PM201, PM206, PM229, and RY2 were found to inhibit the invasion of SV-HUC-1 cells by S. saprophyticus BCRC 10786. To conclude, RY2, PM229, and PM68 strains exhibited inhibitory activity against the urinary tract pathogen S. saprophyticus.

Synergistic antibacterial mechanism of the Lactobacillus crispatus surface layer protein and nisin on Staphylococcus saprophyticus.

SlpB, a surface layer protein isolated from Lactobacillus crispatus, has the potential to enhance the antimicrobial activity of nisin. Previous research indicated that, when combined with nisin, SlpB acted synergistically to inhibit Staphylococcus saprophyticus growth, thus extending the shelf life of chicken meat. In order to understand how SlpB enhances the antibacterial activity of nisin, electron microscopy, confocal laser scanning microscopy, flow cytometry and transmembrane electrical potential analysis were used to study cell wall organization and cell membrane integrity. No remarkable bacteriolytic effects were observed, indicating that cell death could not be attributed to cell lysis, although SlpB caused dramatic modifications of cell wall, thereby altering cell shape. The combination of SlpB and nisin also induced the release of ATP or UV-absorbing materials, as well as sudden dissipation of the transmembrane electrical potential by compromising membrane integrity. Considering that SlpB led to structural disorganization of the cell wall, and nisin access is enhanced to form a stable pore, cell death is a predictable outcome. SlpB significantly enhanced the effect of nisin at half of the minimum inhibitory concentration, which resulted in cell death by destroying the cell wall and cell membrane, therefore providing a new, feasible approach in food preservation.

D-serine transporter in Staphylococcus saprophyticus identified.

Among staphylococci Staphylococcus saprophyticus is the only species that is typically uropathogenic and an important cause of urinary tract infections in young women. The amino acid D-serine occurs in relatively high concentrations in human urine and has a bacteriostatic or toxic effect on many bacteria. In uropathogenic Escherichia coli and S. saprophyticus, the amino acid regulates the expression of virulence factors and can be used as a nutrient. The ability of uropathogens to respond to or to metabolize D-serine has been suggested as a factor that enables colonization of the urinary tract. Until now nothing is known about D-serine transport in S. saprophyticus We generated mutants of putative transporter genes in S. saprophyticus 7108 that show homology to the D-serine transporter cycA of E. coli and tested them in a D-serine depletion assay to analyze the D-serine uptake rate of the cells. The mutant of SPP1070 showed a strong decrease in D-serine uptake. Therefore, SSP1070 was identified as a major D-serine transporter in S. saprophyticus 7108 and was named D-serine transporter A (DstA). D-serine caused a prolonged lag phase of S. saprophyticus in a chemically defined medium. This negative effect was dependent on the presence of DstA.

Subinhibitory antibiotic therapy alters recurrent urinary tract infection pathogenesis through modulation of bacterial virulence and host immunity.

UNLABELLED: The capacity of subinhibitory levels of antibiotics to modulate bacterial virulence in vitro has recently been brought to light, raising concerns over the appropriateness of low-dose therapies, including antibiotic prophylaxis for recurrent urinary tract infection management. However, the mechanisms involved and their relevance in influencing pathogenesis have not been investigated. We characterized the ability of antibiotics to modulate virulence in the uropathogens Staphylococcus saprophyticus and Escherichia coli. Several antibiotics were able to induce the expression of adhesins critical to urothelial colonization, resulting in increased biofilm formation, colonization of murine bladders and kidneys, and promotion of intracellular niche formation. Mice receiving subinhibitory ciprofloxacin treatment were also more susceptible to severe infections and frequent recurrences. A ciprofloxacin prophylaxis model revealed this strategy to be ineffective in reducing recurrences and worsened infection by creating larger intracellular reservoirs at higher frequencies. Our study indicates that certain agents used for antibiotic prophylaxis have the potential to complicate infections. IMPORTANCE: Antibiotics are the mainstay treatment for bacterial infections; however, evidence is emerging that argues these agents may have off-target effects if sublethal concentrations are present. Most studies have focused on changes occurring in vitro, leaving questions regarding the clinical relevance in vivo. We utilized a murine urinary tract infection model to explore the potential impact of low-dose antibiotics on pathogenesis. Using this model, we showed that subinhibitory antibiotics prime uropathogens for adherence and invasion of murine urothelial tissues. These changes in initial colonization promoted the establishment of chronic infection. Furthermore, treatment of chronically infected mice with subtherapeutic ciprofloxacin served to exacerbate infection. A part of these changes was thought to be due to suppression of mucosal immunity, as demonstrated through reductions in cytokine secretion and migration of leukocytes into the urinary tract. This work identifies novel risk factors associated with antibiotic therapy when dosing strategies fall below subtherapeutic levels.

Role of secondary metabolites in establishment of the mutualistic partnership between Xenorhabdus nematophila and the entomopathogenic nematode Steinernema carpocapsae.

Xenorhabdus nematophila engages in a mutualistic partnership with the nematode Steinernema carpocapsae, which invades insects, migrates through the gut, and penetrates into the hemocoel (body cavity). We showed previously that during invasion of Manduca sexta, the gut microbe Staphylococcus saprophyticus appeared transiently in the hemocoel, while Enterococcus faecalis proliferated as X. nematophila became dominant. X. nematophila produces diverse secondary metabolites, including the major water-soluble antimicrobial xenocoumacin. Here, we study the role of X. nematophila antimicrobials in interspecies competition under biologically relevant conditions using strains lacking either xenocoumacin (ΔxcnKL strain), xenocoumacin and the newly discovered antibiotic F (ΔxcnKL:F strain), or all ngrA-derived secondary metabolites (ngrA strain). Competition experiments were performed in Grace's insect medium, which is based on lepidopteran hemolymph. S. saprophyticus was eliminated when inoculated into growing cultures of either the ΔxcnKL strain or ΔxcnKL:F strain but grew in the presence of the ngrA strain, indicating that ngrA-derived antimicrobials, excluding xenocoumacin or antibiotic F, were required to eliminate the competitor. In contrast, S. saprophyticus was eliminated when coinjected into M. sexta with either the ΔxcnKL or ngrA strain, indicating that ngrA-derived antimicrobials were not required to eliminate the competitor in vivo. E. faecalis growth was facilitated when coinjected with either of the mutant strains. Furthermore, nematode reproduction in M. sexta naturally infected with infective juveniles colonized with the ngrA strain was markedly reduced relative to the level of reproduction when infective juveniles were colonized with the wild-type strain. These findings provide new insights into interspecies competition in a host environment and suggest that ngrA-derived compounds serve as signals for in vivo nematode reproduction.

Urinary concentrations and antibacterial activities of nitroxoline at 250 milligrams versus trimethoprim at 200 milligrams against uropathogens in healthy volunteers.

Because of the increasing bacterial resistance of uropathogens against standard antibiotics, such as trimethoprim (TMP), older antimicrobial drugs, such as nitroxoline (NTX), should be reevaluated. This randomized crossover study investigated the urinary concentrations of parent drugs and their metabolites and their antibacterial activities (urinary inhibitory titers [UITs] and urinary bactericidal titers [UBTs]) against uropathogens at three different urinary pH values within 24 h in six healthy volunteers after a single oral dose of NTX at 250 mg versus TMP at 200 mg. In three additional volunteers, urinary bactericidal kinetics (UBK) were studied after oral administration of NTX at 250 mg three times a day. The mean urinary concentrations of NTX and NTX sulfate in 24 h were 0.012 to 0.507 mg/liter and 0.28 to 27.83 mg/liter, respectively. The mean urinary concentrations of TMP were 18.79 to 41.59 mg/liter. The antibacterial activity of NTX was higher in acidic urine than in alkaline urine, and that of TMP was higher in alkaline urine than in acidic urine. The UITs and UBTs of NTX were generally lower than those of TMP except for a TMP-resistant Escherichia coli strain, for which NTX showed higher UITs/UBTs than did TMP. UBK showed mainly bacteriostatic activity of NTX in urine. NTX exhibits mainly bacteriostatic activity and TMP also shows bactericidal activity in urine against susceptible strains. NTX is a more active antibacterial in acidic urine, and TMP is more active in alkaline urine. The cumulative effects of multiple doses or inhibition of bacterial adherence could not be evaluated. (This study has been registered at EudraCT under registration no. 2009-015631-32.).

[Staphylococcus saprophyticus isolated from urine culture in outpatients: epidemiology and antimicrobial susceptibility (Label Bio Elbeuf study - November 2007-July 2009)]. / Staphylococcus saprophyticus isolés d'examens cytobactériologiques urinaires en ville : épidémiologie et sensibilité aux antibiotiques (étude Label Bio Elbeuf - novembre 2007-juillet 2009).

OBJECTIVE: The aim of the study was to determine the distribution and the antibiotic susceptibility of Staphylococcus saprophyticus patterns isolated from urine culture in outpatients (population: 57,000, Elbeuf, Normandie, France). DESIGN: Prospective study from November 2007 to October 2009 in collaboration with three private medical laboratories. Determination of susceptibility to oxacillin by disk diffusion (cefoxitin, and moxalactam), automated method (Vitek BioMérieux 2) and mecA PCR's detection. Determination of the minimum inhibitory concentration by microbroth dilution for other antibiotics. RESULTS: Five thousand and fifty-one bacterial strains isolated, 91 strains of S. saprophyticus (1.8%), 89 in women (2.25%) and two in men (0.18%). S. saprophyticus represented 10.3% and 14.5% of isolates (women respectively aged between 11 and 30; 16 and 20 years); S. saprophyticus is isolated less frequently in winter. mecA PCR detection was positive for two strains. All strains tested were susceptible to ciprofloxacin and furans. Only one strain is resistant to cotrimoxazole. CONCLUSIONS: S. saprophyticus is found mostly in women between 11 to 30 years. Cotrimoxazole (after susceptibility testing) is efficient in case of S. saprophyticus's cystitis. Furans (probabilistic treatment) have to be reevaluated because of the potential for serious adverse effects.

Chemical composition and antibacterial activity of a hexane extract of Tunisian caprifig latex from the unripe fruit of Ficus carica.

CONTEXT: The plant kingdom has become a target in the search for new drugs and biologically active lead compounds. The common Jrani Tunisian caprifig Ficus carica L. (Moraceae) is one of the large number of plant species that are used in folklore medicine yet to be investigated for the treatment of many diseases, including those of infectious nature. OBJECTIVE: Hexane extract of the Tunisian common Jrani caprifig latex was assayed for antibacterial activity against several Gram-positive and Gram-negative bacteria. Chemical composition of the extract was also investigated. MATERIALS AND METHODS: The hexane extract was obtained from Tunisian Jrani caprifig latex by maceration, and then analyzed by gas chromatography-mass spectrometry. The extract was tested in vitro for antibacterial activity by the disc diffusion method and minimal inhibitory concentration (MIC) was also determined for all the test cultures. RESULTS: Thirty-six compounds of the extract were identified, 90.56% of the total area of peaks were coumarins. A strong bactericidal effect was demonstrated. The most sensitive bacteria were Staphylococcus saprophyticus clinical isolate, and Staphylococcus aureus ATCC 25923, with a MIC of 19 µg/mL. DISCUSSION AND CONCLUSION: These findings demonstrate an effective in vitro antibacterial activity of the hexane extract of caprifig latex.

Molecular detection of bacterial pathogens using microparticle enhanced double-stranded DNA probes.

Rapid, specific, and sensitive detection of bacterial pathogens is essential toward clinical management of infectious diseases. Traditional approaches for pathogen detection, however, often require time-intensive bacterial culture and amplification procedures. Herein, a microparticle enhanced double-stranded DNA probe is demonstrated for rapid species-specific detection of bacterial 16S rRNA. In this molecular assay, the binding of the target sequence to the fluorophore conjugated probe thermodynamically displaces the quencher probe and allows the fluorophore to fluoresce. By incorporation of streptavidin-coated microparticles to localize the biotinylated probes, the sensitivity of the assay can be improved by 3 orders of magnitude. The limit of detection of the assay is as few as eight bacteria without target amplification and is highly specific against other common pathogens. Its applicability toward clinical diagnostics is demonstrated by directly identifying bacterial pathogens in urine samples from patients with urinary tract infections.