Rapid Identification Of Bacteria Research Articles

Rapid identification of bacteria with a disposable colorimetric sensing array.

Rapid identification of both species and even specific strains of human pathogenic bacteria grown on standard agar has been achieved from the volatiles they produce using a disposable colorimetric sensor array in a Petri dish imaged with an inexpensive scanner. All 10 strains of bacteria tested, including Enterococcus faecalis and Staphylococcus aureus and their antibiotic-resistant forms, were identified with 98.8% accuracy within 10 h, a clinically important time frame. Furthermore, the colorimetric sensor arrays also proved useful as a simple research tool for the study of bacterial metabolism and as an easy method for the optimization of bacterial production of fine chemicals or other fermentation processes.

Multiplex polymerase chain reaction (PCR) assays for the detection of Enterobacteriaceae in clinical samples

The accurate and rapid identification of bacteria in the enteric tract is necessary for early treatment. In this study, we describeD a novel system which consists of a multiplex polymerase chain reaction (PCR) to simultaneously identify a group of six Enterobacteriaceae members including Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Citrobacter spp., Enterobacter cloacae and Salmonella typhi. Genus and species specific primers were designed for this group of pathogens and conventional multiplex PCR and SYBR green based real time PCR assays were performed to detect these pathogens. All the samples were analysed with a eubacterial real-time PCR assay that enables detection of bacterial DNA and then detection of the organisms was determined using genus and species specific PCR assays. This assay was evaluated using clinical specimens and was found to be quite sensitive and specific. Their PCR results matched with the conventional culture identifications. The conventional and SYBR green real time multiplex PCR assays takes only 3 h to be performed and has the potential to replace the conventional culture technique and thus can speed up the treatment process. This technique has the potential to be a valuable diagnostic tool for simultaneous identification of E. coli, K. pneumoniae, P. mirabilis, Citrobacter spp., E. cloacae and S. typhi.

Rapid Identification of Bacteria from Positive Blood Culture Bottles by Use of Matrix-Assisted Laser Desorption-Ionization Time of Flight Mass Spectrometry Fingerprinting

Early and adequate antimicrobial therapy has been shown to improve the clinical outcome in bloodstream infections (BSI). To provide rapid pathogen identification for targeted treatment, we applied matrix-assisted laser desorption-ionization time of flight (MALDI-TOF) mass spectrometry fingerprinting to bacteria directly recovered from blood culture bottles. A total of 304 aerobic and anaerobic blood cultures, reported positive by a Bactec 9240 system, were subjected in parallel to differential centrifugation with subsequent mass spectrometry fingerprinting and reference identification using established microbiological methods. A representative spectrum of bloodstream pathogens was recovered from 277 samples that grew a single bacterial isolate. Species identification by direct mass spectrometry fingerprinting matched reference identification in 95% of these samples and worked equally well for aerobic and anaerobic culture bottles. Application of commonly used score cutoffs to classify the fingerprinting results led to an identification rate of 87%. Mismatching mostly resulted from insufficient bacterial numbers and preferentially occurred with Gram-positive samples. The respective spectra showed low concordance to database references and were effectively rejected by score thresholds. Spiking experiments and examination of the respective study samples even suggested applicability of the method to mixed cultures. With turnaround times around 100 min, the approach allowed for reliable pathogen identification at the day of blood culture positivity, providing treatment-relevant information within the critical phase of septic illness.

Rapid identification of bacteria and yeast using surface‐enhanced Raman scattering

AbstractSurface‐enhanced Raman scattering (SERS) is a powerful technique used for obtaining chemical information about the molecules and molecular structures in the vicinity of surfaces of noble metal nanostructures. The chemical information acquired through SERS can be used for not only characterization but also detection and identification. In a clinical setting, rapid and accurate identification of micro‐organisms is critical. The biochemical information collected through the SERS spectra can be used for quick identification of micro‐organisms. The concentrated silver colloidal nanoparticles (AgNPs) are simply mixed with micro‐organisms after culturing, and their SERS spectra acquired. Since the nanoparticles are in contact with the cell wall of the micro‐organism, the biochemical information obtained is mostly assumed as originating from the cell wall which the AgNPs are in contact with, and is considered as the ‘fingerprint’ of the micro‐organism, which can be used for the identification. Since a SERS spectrum can be acquired only in seconds, the obtained spectrum can be used for fast micro‐organism identification. The reproducibility of the spectra obtained from micro‐organisms is first tested, and then the obtained spectra are used for the goal. The identification of micro‐organisms in mixtures is also attempted in model mixtures. It is demonstrated that the SERS can be used for fast and accurate identification of micro‐organisms such as bacteria and yeast, even in their mixtures. Four bacteria, i.e. Shigella sonnei, Erwinia amylovara, Proteus vulgaris and DH5α (E. coli strain), and three yeast cells, i.e. Hyphopichia burtonii, Candida parapsilosis and Filobasidiella neoformans are used as model micro‐organisms in the study. Copyright © 2010 John Wiley & Sons, Ltd.

A quick and easy method to identify bacteria by matrix‐assisted laser desorption/ionisation time‐of‐flight mass spectrometry

Concerns with water quality have increased in recent years, in part due to the more frequent contamination of water by pathogens like E. coli and L. pneumophila. Current methods for the typing of bacteria in water samples are based on culture of samples on specific media. These techniques are time-consuming, subject to the impact of interferents and do not totally meet all the requirements of prevention. There is a need for accurate and rapid identification of these microorganisms. This report deals with the detection of bacteria, more precisely of Legionella spp., and the development of an analytical strategy for a rapid and unambiguous identification of these pathogens in water from diverse origins. Therefore, a protein mass mapping using matrix-assisted laser desorption/ionisation mass spectrometry (MALDI MS) of whole bacteria combined with a home-made database of bacteria spectra is applied. A large variety of different bacteria and microorganisms is used to approach the actual composition of samples with numerous interferents. The objective is to propose a universal method for sampling preparation before MALDI MS analysis and optimised spectrometric conditions for reproducible intense peaks. Several experimental factors known to influence signal quality such as time and media of culture have been studied. The proposed method gives promising results for a sure differentiation of Legionella species and subspecies and a rapid identification of bacteria which are the most dangerous or difficult to eradicate. This method is easy to perform with an excellent reproducibility. The analytical protocol and the corresponding database were validated on samples from different origins (cooling tower, plumbing hot water).

A Simple Intact Protein Analysis by MALDI-MS for Characterization of Ribosomal Proteins of Two Genome-Sequenced Lactic Acid Bacteria and Verification of Their Amino Acid Sequences

Rapid identification of bacteria by a bioinformatics-based approach, which processes the mass spectra observed by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS), relies on the calculated masses of ribosomal subunit proteins as biomarkers predicted from amino acid sequences found in protein sequence databases. To verify the actual state of the registered sequence information, a simple intact protein analysis by MALDI-MS using cell lysates as samples was applied to the characterization of ribosomal proteins from genome-sequenced Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus strains. This method avoided the risk of loss of some subunit proteins and the formation of disulfide bonds during the purification of ribosomal proteins. By comparing this with the MALDI mass spectra of different strains and carrying out manual inspection of sequence information, a total of five errors in N-terminal amino acid sequences were identified. After sequence correction, approximately 40 out of 53 subunit proteins could be assigned, considering N-terminal methionine loss only as a post-translational modification. These show promise for use as practical biomarkers for the rapid identification of S. thermophilus and L. bulgaricus. After verification of these amino acid sequences, mass differences relative to those of genome-sequenced strains have the potential for distinguishing bacteria at the strain level.

Rapid identification of bacteria by real-time amplification and sequencing of the 16S rRNA gene

To allow rapid identification of bacteria in pure cultures and blood culture bottles, an assay was developed which is based on real-time amplification and sequencing of bacterial 16 S rRNA genes. In principle, this assay allows identification of bacteria from pure cultures within 6.5 h, and from blood cultures within approximately 7 h.

Development and Evaluation of 16S rDNA Microarray for Detecting Bacterial Pathogens in Cerebrospinal Fluid

The rapid identification of bacteria in cerebrospinal fluid (CSF) is very important for patient management and antimicrobial therapies. We developed a 16S DNA microarray-based method that targets 16S rDNA and can directly detect bacteria from CSF without cultivation. Universal primers and specific probes were designed from the 16S rDNA sequence data retrieved directly from the GenBank database. The specificity of the assay is obtained through a combination of microarray hybridization and enzymatic labeling of the constructed specific probes. Cultivation-dependent assays were used as reference methods in the development and evaluation of the method. With the exception of Mycobacterium tuberculosis and Proteus mirabilis, forty-five positive blood culture media were successfully differentiated. When this procedure was applied directly to 100 CSF specimens, 29 specimens from 16 patients were positive by bacterial culture and 3 culture-positive CSF specimens produced no hybridized signals. The remaining 26 specimens were correctly identified, including one with mixed infection. The accuracy, sensitivity, and specificity of the assay can be increased further by designing more oligonucleotides for the microarray. This method is versatile and makes it possible to detect more bacteria in a single assay and discriminate different bacterial genera.

Accuracy of identification and susceptibility results by direct inoculation of Vitek 2 cards from positive BACTEC cultures

Rapid identification of bacteria and prompt acquisition of susceptibility results are valuable for patient care. The objective of the present study was to determine the accuracy of direct inoculation of Vitek 2 cards from positive BACTEC cultures compared to inoculation of the cards from subculture plates. Positive BACTEC cultures sampled between March 2001 and June 2002 were included. The results of direct inoculation were compared with the results of inoculation of Vitek 2 cards from subcultures. Of 161 gram-negative bacilli, 129 (80%) were correctly identified by direct inoculation compared to 145 of 161 (90%) by subculture. Susceptibility testing was performed on 2,862 antibiotic-isolate combinations. The essential agreement was 98.7%. The number of very major, major, and minor errors was 1 (0.2% of resistant strains), 1 (0.04% of susceptible strains), and 68, respectively. Direct identification of Staphylococcus spp. was not performed, but antimicrobial susceptibility was tested using 6,042 antibiotic-isolate combinations. The essential agreement was 95.2%. The number of very major, major, and minor errors was 73 (4.5% of resistant strains), 32 (0.8% of susceptible strains), and 106, respectively. Eighty-four percent of the very major errors occurred with trimethoprim-sulfamethoxazole. The results show that direct inoculation of Vitek cards is valuable as a rapid routine method for identification and susceptibility testing of gram-negative bacilli. For Staphylococcus spp., the susceptibility results obtained after direct inoculation of Vitek 2 cards are also acceptable except for those obtained with trimethoprim-sulfamethoxazole. Susceptibility results for this antibiotic, if obtained using direct inoculation, should not be reported to the clinician.

Rapid identification of bacteria from positive blood cultures by terminal restriction fragment length polymorphism profile analysis of the 16S rRNA gene.

Bacteremia results in significant morbidity and mortality, especially among patient populations that are immunocompromised. Broad-spectrum antibiotics are administered to patients suspected to have bloodstream infections that are awaiting diagnosis that depends on blood culture analysis. Significant delays in identification of pathogens can result, primarily due to the dependence on growth-based identification systems. To address these limitations, we took advantage of terminal restriction fragment (TRF) length polymorphisms (T-RFLP) due to 16S ribosomal DNA (rDNA) sequence diversity to rapidly identify bacterial pathogens directly from positive blood culture. TRF profiles for each organism were determined by sizing fragments from restriction digests of PCR products derived from two sets of 16S rDNA-specific fluorescent dye-labeled primers. In addition, we created a TRF profile database (TRFPD) with 5899 predicted TRF profiles from sequence information representing 2860 different bacterial species. TRF profiles were experimentally determined for 69 reference organisms and 32 clinical isolates and then compared against the predicted profiles in the TRFPD. The predictive value of the profiles was found to be accurate to the species level with most organisms tested. In addition, identification of 10 different genera was possible with profiles comprising two or three TRFs. Although it was possible to identify Enterobacteriaceae by using a profile of three TRFs, the similarity of the TRF profiles of these organisms makes differentiation of species less reliable with the current method. The ability to rapidly (i.e., within approximately 8 h) identify bacteria from blood cultures has potential for reducing unnecessary use of broad-spectrum antibiotics and promoting more timely prescription of appropriate antibiotics.

Evaluation of the Vitek 2 system for rapid identification of clinical isolates of gram-negative bacilli and members of the family Streptococcaceae.

Accuracy of the Vitek 2 automated system (bioMérieux Vitek, USA) for rapid identification of bacteria was evaluated using a collection of 858 epidemiologically unrelated gram-negative and 99 gram-positive clinical isolates. Isolates were tested after subculturing to ensure purity. Conventional agar-based biochemical tests (Steers replicator) were used as a reference method of identification. Gram-negative bacteria were identified to the species level with 95.3% accuracy by the system ( Enterobacteriaceae, 95.9%; and non- Enterobacteriaceae, 92.5%), and gram-positive isolates with 72% accuracy. Although Vitek 2 identified routine clinical isolates of gram-negative bacilli and Enterococcus faecalis and Enterococcus faecium reliably, rapidly, and reproducibly, improvement is required in the identification of less common species of enterococci and viridans group streptococci.

Rapid diagnosis of bacteremia by universal amplification of 23S ribosomal DNA followed by hybridization to an oligonucleotide array.

The rapid identification of bacteria in blood cultures and other clinical specimens is important for patient management and antimicrobial therapy. We describe a rapid (<4 h) detection and identification system that uses universal PCR primers to amplify a variable region of bacterial 23S ribosomal DNA, followed by reverse hybridization of the products to a panel of oligonucleotides. This procedure was successful in discriminating a range of bacteria in pure cultures. When this procedure was applied directly to 158 unselected positive blood culture broths on the day when growth was detected, 125 (79.7%) were correctly identified, including 4 with mixed cultures. Nine (7.2%) yielded bacteria for which no oligonucleotide targets were present in the oligonucleotide panel, and 16 culture-positive broths (10.3%) produced no PCR product. In seven of the remaining eight broths, streptococci were identified but not subsequently grown, and one isolate of Staphylococcus aureus was misidentified as a coagulase-negative staphylococcus. The accuracy, range, and discriminatory power of the assay can be continually extended by adding further oligonucleotides to the panel without significantly increasing complexity or cost.

Rapid identification of bacteria in blood cultures by using fluorescently labeled oligonucleotide probes.

The applicability of whole-cell hybridization for the identification of pathogenic bacteria in blood from septic patients was examined. Oligonucleotide probes, fluorescently labeled with fluorescein isothiocyanate, directed against the variable regions of the 16S rRNAs of the following bacterial species and/or genera were used: Streptococcus spp., Enterococcus faecalis, Staphylococcus aureus, coagulase-negative staphylococci (CoNS), Escherichia coli, Pseudomonas aeruginosa, and the Enterobacteriaceae family. A probe specific for the rRNAs of almost all bacteria and its complementary, reversed counterpart was used as positive and negative control, respectively. The probes were used in conjunction with a fast and simple-to-use protocol for whole-cell hybridization. This protocol yields an identification after 25 to 45 min, depending on whether the bacterium is gram positive or gram negative. A total of 182 blood samples which tested positive in a blood culture machine were investigated. All probes except for the ones for S. aureus and the CoNS showed sensitivities and specificities of 1.000. It was concluded that whole-cell hybridization is well suited for the fast screening of septic blood containing streptococci and/or enterococci or gram-negative rods.

Rapid identification of bacteria from positive blood cultures by fluorescence-based PCR-single-strand conformation polymorphism analysis of the 16S rRNA gene.

Bacteremia continues to result in significant morbidity and mortality, particularly in patients who are immunocompromised. Currently, patients with suspected bacteremia are empirically administered broad-spectrum antibiotics, as definitive diagnosis relies upon the use of blood cultures, which impose significant delays in and limitations to pathogen identification. To address the limitations of growth-based identification, the sequence variability of the 16S rRNA gene of bacteria was targeted for rapid identification of bacterial pathogens isolated directly from blood cultures using a fluorescence-based PCR-single-strand conformation polymorphism (SSCP) protocol. Species-specific SSCP patterns were determined for 25 of the most common bacterial species isolated from blood cultures; these isolates subsequently served as a reference collection for bacterial identification for new cases of bacteremia. A total of 272 blood-culture-positive patient specimens containing bacteria were tested. A previously determined SSCP pattern was observed for 251 (92%) specimens, with 21 (8%) specimens demonstrating SSCP patterns distinct from those in the reference collection. Time to identification from blood culture positivity ranged from 1 to 8 days with biochemical testing, whereas identification by fluorescence-based capillary electrophoresis was obtained as early as 7 h at a calculated cost of $10 (U.S. currency) per specimen when tested in batches of 10. Limitations encountered included the inability to consistently detect mixed cultures as well as some species demonstrating identical SSCP patterns. This method can be applied directly to blood cultures or whole-blood specimens, where early pathogen identification would result in a timely diagnosis with possible implications for patient management costs and the mortality and morbidity of infections.

Abbreviated, rapid identification of bacteria and yeasts

Summary As described above, many alternative ways to rapidly identify organisms commonly seen in clinical specimens are available. While most reagents can be purchased, large laboratories may find it cost-effective to make a few. Examples of these reagents are summarized in Table 3. Use of rapid spot-test methods is at the discretion of each laboratory, but clearly a large amount of time and resources can be saved and patient care will benefit from the rapid reporting of organism identifications.

Rapid identification of bacteria by direct matrix-assisted laser desorption/ionization mass spectrometric analysis of whole cells.

Several characteristic ions were observed during the direct analysis of a variety of both gram-negative and gram-positive intact bacterial cells by the matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) technique. The entire process, involving absolutely no sample processing, could be completed in less than ten minutes. A number of specific biomarkers, generated reproducibly for each type of cell from the corresponding mass spectrum, permitted the identification, as well as the distinction, of pathogenic bacteria from their non-pathogenic counterparts. In addition, individual strains of a specific organism could also be differentiated easily. Some of these biomarkers correspond to those observed earlier during the MALDI-MS analysis of protein extracts of the same bacteria. This approach, which can yield valuable data for rapid classification and detection of microorganisms, represents a substantial breakthrough for rapid screening of environmental as well as biological samples.

Rapid identification of bacteria by PCR-single-strand conformation polymorphism.

A new molecular biological approach for the identification of bacteria is described. This approach employs PCR of bacterial cell lysates with conserved primers located in the 16S rRNA sequence flanking a variable region, and analysis of the amplified product was based on the principle of single-strand conformation polymorphism (SSCP). The PCR product was denatured and separated on a nondenaturing polyacrylamide gel. SSCP patterns were detected by silver staining the nucleic acids. The mobility of the single-stranded DNA is sequence dependent and could be used to identify the unknown bacteria. Feasibility of the technique was demonstrated for a broad panel of gram-negative and gram-positive bacteria. We tested over 100 strains of bacteria representing 15 genera and 40 species. With the use of only two primer sets, P11P-P13P and ER10-ER11, we were capable to discriminate the tested species at the genus and species levels. Species-specific patterns were obtained for, e.g., Clostridium spp., Listeria spp., Pseudomonas spp., and Enterobacter spp. PCR-SSCP is a sensitive technique; e.g., the sensitivity obtained for Escherichia coli cells was 30 CFU. This technique is a simple and rapid method for the detection and identification of a wide spectrum of bacteria by whole-cell-based PCR amplification with the use of conserved primers and identification by nondenaturing gel electrophoresis.

Rapid identification of bacteria on the basis of polymerase chain reaction-amplified ribosomal DNA spacer polymorphisms.

To facilitate genus and species level identification of a broad range of bacteria without the requirement of presumptive identification, we have developed a unified set of primers and polymerase chain reaction conditions to amplify spacer regions between the 16S and 23S genes in the prokaryotic rRNA genetic loci. Spacer regions within these loci show a significant level of length and sequence polymorphism across both genus and species lines. A generic pair of priming sequences was selected for the amplification of these polymorphisms from highly conserved sequences in the 16S and 23S genes occurring adjacent to these polymorphic regions. This single set of primers and reaction conditions was used for the amplification of 16S-23S spacer regions for over 300 strains of bacteria belonging to eight genera and 28 species or serotypes, including Listeria, Staphylococcus, and Salmonella species and additional species related to these pathogenic organisms. When the spacer amplification products were resolved by electrophoresis, the resulting patterns could be used to distinguish all of the species of bacteria within the test group. Unique elements in the amplification product patterns generally clustered at the species level, although some genus-specific characteristics were also observed. On the basis of the results obtained with our test group of 300 bacterial strains, amplification of the 16S-23S ribosomal spacer region is a suitable process for generating a data base for use in a polymerase chain reaction-based identification method, which can be comprehensively applied to the bacterial kingdom.

Availability of 6-hydroxynicotinic acid for rapid identification of pseudomonas aeruginosa and serratia marcescens

Gas chromatography—mass spectrometry has been used to identify specific metabolites produced by Gram-negative bacteria such as Pseudomonas aeruginosa, Serratia marcescens, Klebsiella pneumoniae and Escherichia coli in a defined medium. 6-Hydroxynicotinic acid was detected in spent culture media of Pseudomonas aeruginosa and Serratia marcescens, but could not be detected in those of Klebsiella pneumoniae and Escherichia coli. The production of 6-hydroxynicotinic acid was recognized by the addition of nicotinic acid in urine with Pseudomonas aeruginosa or Serratia marcescens, but not without the addition of nicotinic acid. Among 10 5 Pseudomonas aeruginosa per 1 ml of urine (criteria for the diagnosis of urinary tract infection), 0.15 μg of 6-hydroxynicotinic acid was detected in urine at 4 h incubation with nicotinic acid at the optimum pH of 6.9, 38°C. The production of 6-hydroxynicotinic acid was proportional to the number of the bacteria and displayed a time dependency. These results suggest that the availability of 6-hydroxynicotinic acid might make for more rapid identification of bacteria than current methods.

Automated immunofluorescent speciation of oral bacteria using flow cytometry.

Mixtures containing two bacterial species were analyzed using flow cytometric techniques. Light scattering characteristics of Streptococcus mutans and Actinomyces viscosus show unique profiles for pure cultures. However, the light scatter analysis of a mixture containing these two species demonstrates overlapping near the origin. Thus, light scatter analysis was not sufficient to speciate bacteria with different morphologies. Labeling of samples with species-specific immunofluorescent antibodies permitted speciation of mixtures. As the percentage of the bacterium to which the antibody is directed increased in a two-component mixture, fluorescent flow cytometric analysis showed a corresponding increase in the percentage of cells displaying fluorescent labeling. These methods could permit the rapid identification of bacteria from oral sites without culturing.