Rapid Identification Of Bacteria Research Articles

Evaluation of a micro/nanofluidic chip platform for the high-throughput detection of bacteria and their antibiotic resistance genes in post-neurosurgical meningitis

BackgroundPost-neurosurgical meningitis (PNM) is one of the most severe hospital-acquired infections worldwide, and a large number of pathogens, especially those possessing multi-resistance genes, are related to these infections. Existing methods for detecting bacteria and measuring their response to antibiotics lack sensitivity and stability, and laboratory-based detection methods are inconvenient, requiring at least 24h to complete. Rapid identification of bacteria and the determination of their susceptibility to antibiotics are urgently needed, in order to combat the emergence of multi-resistant bacterial strains. MethodsThis study evaluated a novel, fast, and easy-to-use micro/nanofluidic chip platform (MNCP), which overcomes the difficulties of diagnosing bacterial infections in neurosurgery. This platform can identify 10 genus or species targets and 13 genetic resistance determinants within 1h, and it is very simple to operate.A total of 108 bacterium-containing cerebrospinal fluid (CSF) cultures were tested using the MNCP for the identification of bacteria and determinants of genetic resistance. The results were compared to those obtained with conventional identification and antimicrobial susceptibility testing methods. ResultsFor the 108 CSF cultures, the concordance rate between the MNCP and the conventional identification method was 94.44%; six species attained 100% consistency. For the production of carbapenemase- and extended-spectrum beta-lactamase (ESBL)-related antibiotic resistance genes, both the sensitivity and specificity of the MNCP tests were high (>90.0%) and could fully meet the requirements of clinical diagnosis. ConclusionsThe MNCP is fast, accurate, and easy to use, and has great clinical potential in the treatment of post-neurosurgical meningitis.

Buckyballs conjugated with nucleic acid sequences identifies microorganisms in live cell assays

BackgroundRapid identification of bacteria can play an important role at the point of care, evaluating the health of the ecosystem, and discovering spatiotemporal distributions of a bacterial community. We introduce a method for rapid identification of bacteria in live cell assays based on cargo delivery of a nucleic acid sequence and demonstrate how a mixed culture can be differentiated using a simple microfluidic system.MethodsC60 Buckyballs are functionalized with nucleic acid sequences and a fluorescent reporter to show that a diversity of microorganisms can be detected and identified in live cell assays. The nucleic acid complexes include an RNA detector, targeting a species-specific sequence in the 16S rRNA, and a complementary DNA with an attached fluorescent reporter. As a result, each bacterium can be detected and visualized at a specific emission frequency through fluorescence microscopy.ResultsThe C60 probe complexes can detect and identify a diversity of microorganisms that include gram-position and negative bacteria, yeast, and fungi. More specifically, nucleic-acid probes are designed to identify mixed cultures of Bacillus subtilis and Streptococcus sanguinis, or Bacillus subtilis and Pseudomonas aeruginosa. The efficiency, cross talk, and accuracy for the C60 probe complexes are reported. Finally, to demonstrate that mixed cultures can be separated, a microfluidic system is designed that connects a single source-well to multiple sinks wells, where chemo-attractants are placed in the sink wells. The microfluidic system allows for differentiating a mixed culture.ConclusionsThe technology allows profiling of bacteria composition, at a very low cost, for field studies and point of care.

The use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for rapid bacterial identification in patients with smear-positive bacterial meningitis

ObjectivesTo assess the potential of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) in rapid identification of bacteria from smear-positive cerebrospinal fluid (CSF) in a cohort of patients with meningitis. MethodsSingle-centre observational study, including adults and children with community-acquired or postneurosurgical bacterial meningitis. Meningitis was defined using established criteria. Samples of CSF that had a positive CSF Gram stain were directly examined by MALDI-TOF-MS. Identification was considered accurate when identical to the CSF culture or PCR results (species and genus level). Laboratory workers performing the MALDI-TOF-MS and interpreting its results were blinded to the direct smear results, except for the fact that it was positive. MALDI-TOF-MS results were not conveyed to clinicians. ResultsMALDI-TOF-MS was tested on 44 CSF samples; ten samples were obtained from patients with community-acquired meningitis, and 34 samples were from patients with postneurosurgical meningitis. The assay identified bacteria correctly in 17/21 of the samples with Gram-negative rods observed on the direct smear, all obtained from patients who had undergone neurosurgery, (sensitivity 81%, 95% CI 64.2%–97.7%). In the postneurosurgical group, Gram-positive cocci were identified correctly in only 1/11 (9.1%) of the samples, and Candida species were not identified in two samples. Among patients with community-acquired meningitis, the assay did not identify Streptococcus pneumoniae in eight of eight samples, Neisseria meningitidis in one sample (1/1), and Streptococcus agalactiae in one sample (1/1). ConclusionsWe found MALDI-TOF-MS to be useful in the rapid identification of Gram-negative rods directly from smear-positive CSF samples, but not of Gram-positive bacteria.

Reprint of “Application of next generation sequencing in clinical microbiology and infection prevention”

Current molecular diagnostics of human pathogens provide limited information that is often not sufficient for outbreak and transmission investigation. Next generation sequencing (NGS) determines the DNA sequence of a complete bacterial genome in a single sequence run, and from these data, information on resistance and virulence, as well as information for typing is obtained, useful for outbreak investigation. The obtained genome data can be further used for the development of an outbreak-specific screening test. In this review, a general introduction to NGS is presented, including the library preparation and the major characteristics of the most common NGS platforms, such as the MiSeq (Illumina) and the Ion PGM™ (ThermoFisher). An overview of the software used for NGS data analyses used at the medical microbiology diagnostic laboratory in the University Medical Center Groningen in The Netherlands is given. Furthermore, applications of NGS in the clinical setting are described, such as outbreak management, molecular case finding, characterization and surveillance of pathogens, rapid identification of bacteria using the 16S-23S rRNA region, taxonomy, metagenomics approaches on clinical samples, and the determination of the transmission of zoonotic micro-organisms from animals to humans. Finally, we share our vision on the use of NGS in personalised microbiology in the near future, pointing out specific requirements.

Evaluation of three sample preparation methods for the direct identification of bacteria in positive blood cultures by MALDI-TOF

BackgroundPatient mortality is significantly reduced by rapid identification of bacteria from sterile sites. MALDI-TOF can identify bacteria directly from positive blood cultures and multiple sample preparation methods are available. We evaluated three sample preparation methods and two MALDI-TOF score cut-off values. Positive blood culture bottles with organisms present in Gram stains were prospectively analysed by MALDI-TOF. Three lysis reagents (Saponin, SDS, and SepsiTyper lysis bufer) were applied to each positive culture followed by centrifugation, washing and protein extraction steps. Methods were compared using the McNemar test and 16S rDNA sequencing was used to assess discordant results.ResultsIn 144 monomicrobial cultures, using ≥2.000 as the cut-off value, species level identifications were obtained from 69/144 (48%) samples using Saponin, 86/144 (60%) using SDS, and 91/144 (63%) using SepsiTyper. The difference between SDS and SepsiTyper was not statistically significant (P = 0.228). Differences between Saponin and the other two reagents were significant (P < 0.01). Using ≥1.700 plus top three results matching as the cut-off value, species level identifications were obtained from 100/144 (69%) samples using Saponin, 103/144 (72%) using SDS, and 106/144 (74%) using SepsiTyper and there was no statistical difference between the methods. No true discordances between culture and direct MALDI-TOF identification were observed in monomicrobial cultures. In 32 polymicrobial cultures, MALDI-TOF identified one organism in 34–75% of samples depending on the method.ConclusionsThis study demonstrates two inexpensive in-house detergent lysis methods are non-inferior to a commercial kit for analysis of positive blood cultures by direct MALDI-TOF in a clinical diagnostic microbiology laboratory.

Application of next generation sequencing in clinical microbiology and infection prevention

Current molecular diagnostics of human pathogens provide limited information that is often not sufficient for outbreak and transmission investigation. Next generation sequencing (NGS) determines the DNA sequence of a complete bacterial genome in a single sequence run, and from these data, information on resistance and virulence, as well as information for typing is obtained, useful for outbreak investigation. The obtained genome data can be further used for the development of an outbreak-specific screening test. In this review, a general introduction to NGS is presented, including the library preparation and the major characteristics of the most common NGS platforms, such as the MiSeq (Illumina) and the Ion PGM™ (ThermoFisher). An overview of the software used for NGS data analyses used at the medical microbiology diagnostic laboratory in the University Medical Center Groningen in The Netherlands is given. Furthermore, applications of NGS in the clinical setting are described, such as outbreak management, molecular case finding, characterization and surveillance of pathogens, rapid identification of bacteria using the 16S-23S rRNA region, taxonomy, metagenomics approaches on clinical samples, and the determination of the transmission of zoonotic micro-organisms from animals to humans. Finally, we share our vision on the use of NGS in personalised microbiology in the near future, pointing out specific requirements.

Rapid bacteria identification from environmental mining samples using MALDI-TOF MS analysis.

Copper mining has polluted soils and water, causing a reduction of the microbial diversity and a change in the structure of the resident bacterial communities. In this work, selective isolation combined with MALDI-TOF MS and the 16S rDNA method were used for characterizing cultivable bacterial communities from copper mining samples. The results revealed that MALDI-TOF MS analysis can be considered a reliable and fast tool for identifying copper-resistant bacteria from environmental samples at the genera level. Even though some results were ambiguous, accuracy can be improved by enhancing reference databases. Therefore, mass spectra analysis provides a reliable method to facilitate monitoring of the microbiota from copper-polluted sites. The understanding of the microbial community diversity in copper-contaminated sites can be helpful to understand the impact of the metal on the microbiome and to design bioremediation processes.

Aplicación de la espectrometría de masas en la identificación de bacterias

Correct and rapid identification of bacteria is essential for the correct diagnosis and treatment of infected patients. Until a few years ago, biochemical, colorimetric or even antibiotic sensitivity tests were used to identify genera and species. The main limitations of these methods were the time needed for their performance and the difficulty of distinguishing between microorganisms that were little reactive, highly similar, or difficult to culture. Many of these problems have been solved by the introduction of mass spectrometry (MS) in the laboratory with the use of MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight). Knowledge of the strengths and weaknesses of this technology is essential to be able to take maximum advantage of this technique. Not all microorganisms can be identified with the same ease and reliability by MALDI-TOF and microbiologists need to know how to interpret the results obtained with this technique and the available alternatives in order to identify the microorganisms causing the most problems. This article aims to summarise the available information on the correct identification of the main human pathogenic bacteria through the use of MALDI-TOF MS, focusing on Gram-negative, Grampositive and anaerobic microorganisms. The main factors that must be taken into account for the reliable identification of any bacterium are the conditions for culture, sample preparation with the ideal extraction method and especially the use of a correct and updated database.

Rapid Bacteria Identification Using Multivariate Classifier on Diabetic Foot Infection

Rapid Bacteria Identification Using Multivariate Classifier on Diabetic Foot Infection

Rapid identification of bacteria in positive blood culture by chromogenic culture media

Rapid identification of bacteria in positive blood culture by chromogenic culture media

Nonculture techniques for the detection of bacteremia and fungemia.

Bacteremia and fungemia account for a substantial proportion of all cases of severe sepsis. Antibiotic resistance is a contributing factor in many hospital-acquired infection deaths. Traditional phenotypic methods for the identification of bacteria and yeasts from positive blood cultures and determining antimicrobial susceptibility require 48-72 h, delaying optimal therapy and negatively impacting patient outcomes. Molecular methods, including nonamplified DNA probe panels and peptide nucleic acid probes, and nucleic acid amplification methods such as PCR, proteomic methods (matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry) and direct biochemical tests provide more rapid identification of bacteria and fungi, and in some cases antimicrobial resistance markers, from positive blood cultures, as well as directly from whole blood. These methods vary in the breadth of organisms that they detect, and equally important, their ease of use. This article examines the principles, performance and practicality of the various rapid, nonculture techniques for the detection of bacteremia and fungemia.

Rapid Identification of Bacteria and Candida Pathogens in Peritoneal Dialysis Effluent from Patients with Peritoneal Dialysis-Related Peritonitis by Use of Multilocus PCR Coupled with Electrospray Ionization Mass Spectrometry

PCR coupled with electrospray ionization mass spectrometry (PCR/ESI-MS) was compared with culture for pathogen detection in peritoneal dialysis (PD)-related peritonitis. Of 21 samples of PD effluent, PCR/ESI-MS identified microorganisms in 18 (86%) samples, including Mycobacterium tuberculosis in 1 culture-negative sample. Of 15 double-positive samples, PCR/ESI-MS and culture reached levels of agreement of 100% (15/15) and 87.5% (7/8) at the genus and species levels, respectively. PCR/ESI-MS can be used for rapid pathogen detection in PD-related peritonitis.

Rapid Identification of Microbes in Positive Blood Cultures by Use of the Vitek MS Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry System

Sepsis is a major cause of death worldwide among nonhospitalized people and hospitalized patients. A wide range of pathogens are involved, and the correct identification and correct antimicrobial therapy are critical to ensure optimal clinical outcomes. With the recent introduction of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), rapid identification of bacteria and fungi is now possible. The purpose of this study was to develop a rapid technique for identifying organisms in positive blood cultures using the Vitek MS system (bioMérieux). This technique is a lysis centrifugation method which involves a four-step washing and centrifugation procedure. A total of 253 positive monomicrobial blood cultures (Bactec Plus aerobic, anaerobic, and pediatric bottles) were tested using the Vitek MS system (KnowledgeBase version 2.0), with 92.1% and 88.1% of organisms overall being identified to the genus level and the species level, respectively. Of 161 Gram-positive bacterial isolates, 95.7% and 90.1% were identified to the genus level and the species level, respectively; of 92 Gram-negative bacterial isolates, 84.7% and 83.7% were identified to the genus level and the species level, respectively. The results obtained using this method demonstrate that the Vitek MS system can be used for rapid and effective identification of bacteria from positive blood cultures within 30 to 45 min after the positive signal has been provided by the Bactec FX blood culture system (Becton, Dickinson). This will lead to faster administration of the appropriate antimicrobial therapy and increase the chances for optimal clinical outcomes for patients.

Rapid identification of bacteria from bioMerieux BacT/ALERT blood culture bottles by MALDI-TOF MS

Several studies have reported poor results when trying to identify microorganisms directly from the bioMerieux BacT/ALERT blood culture system using matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF) mass spectrometry. The aim of this study is to evaluate two new methods, Sepsityper and an enrichment method for direct identification of microorganisms from this system. For both methods the samples were processed using the Bruker Microflex LT mass spectrometer (Biotyper) using the Microflex Control software to obtain spectra. The results from direct analysis were compared with those obtained by subculture and subsequent identification. A total of 350 positive blood cultures were processed simultaneously by the two methods. Fifty-three cultures were polymocrobial or failed to grow any organism on subculture, and these results were not included as there was either no subculture result, or for polymicrobial cultures it was known that the Biotyper would not be able to distinguish the constituent organisms correctly. Overall, the results showed that, contrary to previous reports, it is possible to identify bacteria directly from bioMerieux blood culture bottles, as 219/297 (74%) correct identifications were obtained using the Bruker Sepsityper method and 228/297 (77%) were obtained for the enrichment method when there is only one organism was present. Although the enrichment method was simpler, the reagent costs for the Sepsityper method were approximately £4.00 per sample compared to £0.50. An even simpler and cheaper method, which was less labour-intensive and did not require further reagents, was investigated. Seventy-seven specimens from positive signalled blood cultures were analysed by inoculating prewarmed blood agar plates and analysing any growth after 1-, 2-and 4-h periods of incubation at 37° C, by either direct transfer or alcohol extraction. This method gave the highest number of correct identifications, 66/77 (86%), and was cheaper and less labour-intensive than either of the two above methods.

Rapid identification of bacteria and candida using pna-fish from blood and peritoneal fluid cultures: a retrospective clinical study

BackgroundPeptide nucleic acid fluorescent in situ hybridization (PNA-FISH) is a rapid and established method for identification of Candida sp., Gram positive, and Gram negative bacteria from positive blood cultures. This study reports clinical experience in the evaluation of 103 positive blood cultures and 17 positive peritoneal fluid cultures from 120 patients using PNA-FISH. Our study provides evidence as to potential pharmaceutical cost savings based on rapid pathogen identification, in addition to the novel application of PNA-FISH to peritoneal fluid specimens.MethodsIdentification accuracy and elapsed time to identification of Gram positives, Gram negatives, and Candida sp., isolated from blood and peritoneal fluid cultures were assessed using PNA-FISH (AdvanDx), as compared to standard culture methods. Patient charts were reviewed to extrapolate potential pharmaceutical cost savings due to adjustment of antimicrobial or antifungal therapy, based on identification by PNA-FISH.ResultsIn blood cultures, time to identification by standard culture methods for bacteria and Candida sp., averaged 83.6 hours (95% CI 56.7 to 110.5). Identification by PNA-FISH averaged 11.2 hours (95% CI 4.8 to 17.6). Overall PNA-FISH identification accuracy was 98.8% (83/84, 95% CI 93.5% to 99.9%) as compared to culture. In peritoneal fluid, identification of bacteria by culture averaged 87.4 hours (95% CI −92.4 to 267.1). Identification by PNA-FISH averaged 16.4 hours (95% CI −57.3 to 90.0). Overall PNA-FISH identification accuracy was 100% (13/13, 95% CI 75.3% to 100%). For Candida sp., pharmaceutical cost savings based on PNA-FISH identification could be $377.74/day. For coagulase-negative staphylococcus (CoNS), discontinuation of vancomycin could result in savings of $20.00/day.ConclusionsIn this retrospective study, excellent accuracy of PNA-FISH in blood and peritoneal fluids with reduced time to identification was observed, as compared to conventional culture-based techniques. Species-level identification based on PNA-FISH could contribute to notable cost savings due to adjustments in empiric antimicrobial or antifungal therapy as appropriate to the pathogen identified.

Matrix-assisted laser desorption/ionisation-time of flight mass spectrometry: rapid identification of bacteria isolated from patients with cystic fibrosis

Despite extensive research into the diagnosis and management of cystic fibrosis (CF) over the past decades, sufferers still have a median life expectancy of less than 37 years. Respiratory tract infections have a significant role in increasing the morbidity and mortality of patients with CF via a progressive decline in lung function. Rapid identification of organisms recovered from CF sputum is necessary for effective management of respiratory tract infections; however, standard techniques of identification are slow, technically demanding and expensive. The aim of this study is to asses the suitability of matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS) in identifying bacteria isolated from the respiratory tract of patients with CF, and is assessed by testing the accuracy of MALDI-TOF MS in identifying samples from a reference collection of rare CF strains in conjunction with comparing MALDI-TOF MS and standard techniques in identifying clinical isolates from sputum samples of CF patients. MALDI-TOF MS accurately identified 100% of isolates from the reference collection of rare CF pathogens (EuroCare CF collection). The isolate identification given by MALDI-TOF MS agreed with that given by standard techniques for 479/481 (99.6%) clinical isolates obtained from respiratory samples provided by patients with CF In two (0.4%) of 481 samples there was a discrepancy in identification between MALDI-TOF MS and standard techniques. One organism was identified as Pseudomonas aeruginosa by MALDI-TOF but could only be identified by the laboratory’s standard methods as of the Pseudomonas genus. The second organism was identified as P. beteli by MALDI-TOF MS and Stenotrophomonas maltophilia by standard methods. This study shows that MALDI-TOF MS is superior to standard techniques in providing cheap, rapid and accurate identification of CF sputum isolates.

Quantitative evaluation of periprosthetic infection by real-time polymerase chain reaction: a comparison with conventional methods

Several recent studies have demonstrated the limited accuracy of conventional culture methods for diagnosing periprosthetic infections. We have applied real-time polymerase chain reaction (PCR) assays for the rapid identification of bacteria around implants and reported its utility. However, the capability of quantification is also a useful feature of this type of assay. The aim of our study was to validate the usefulness of quantitative analyses using real-time PCR of cases with clinical periprosthetic infections in comparison with more established tests, such as C-reactive protein (CRP) levels, microbiologic cultures, and histopathology. Fifty-six joints with suspected infections were reviewed retrospectively. A universal PCR assay was used to perform the quantitative analyses. The differences in the threshold cycles between clinical samples and a negative control (∆Ct) in each case were calculated. The results of the quantitative PCR assay were compared with CRP levels, microbiologic cultures, and histopathology. There was a significant correlation found between the CRP and ∆Ct values. There were also significant differences found in the ∆Ct values according to CRP levels, with higher CRP levels showing higher ∆Ct values. Similarly, there were significant differences in the ∆Ct measurements in our culture results and among our pathologic evaluations. We confirmed that quantification by universal PCR based on the ∆Ct correlated with preoperative CRP levels and was associated with the microbiologic culture results and pathologic severity. This quantification method may be valuable for assessing infection severity.

Rapid lipid profiling of bacteria by online MALDI-TOF mass spectrometry

The rapid identification of bacteria is of increasing interest in food, biosafety, and clinical analysis. In this paper, we report a technique for rapid lipid profiling of bacteria by online droplet-deposited MALDI mass spectrometry. In the experiment, lipids in bacterial cells (Escherichia coli, Salmonella, and Bacillus subtilis) are extracted with the mixture of chloroform/methanol via ultrasonication. The extracts are nebulized into suspended droplets and then instantly analyzed with an online droplet-deposited MALDI-TOF mass spectrometer. The lipid profiles of the three bacteria cultured in nutrient broth and LB (Lysogeny Broth) medium are obtained. The lipids phosphatidylethanolamine (PE), diglycosyl diacylglycerol (DGDG), and phosphatidylglycerol (PG) are observed in the MALDI-TOF mass spectra. The MALDI-TOF mass spectrum of each bacterial extract shows a characteristic profile. The experimental results demonstrate that the present technique is capable of conducting rapid lipid analysis and has the potential for rapid bioaerosol detection.

Citations

Citations

Rapid identification of bacteria in positive blood culture by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Blood culture is probably the most significant specimen used for the diagnosis of bacterial infections, especially for bloodstream infections. In the present study, we compared the resin-containing BD BACTEC™ Plus-Aerobic (Becton Dickinson), non-charcoal-containing BacT/Alert(®) SA (bioMérieux), and charcoal-containing BacT/Alert(®) FA (bioMérieux) blood culture bottles with direct identification by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). A total of 103 bacterial isolates, from clinical blood cultures, representing the most frequent 13 genera and 24 species were examined. Bacteria were extracted from positive blood culture broth by density centrifugation and then subjected to identification by MALDI-TOF MS using two different volumes and chemical treatments. Overall, correct identification by MALDI-TOF MS was obtained for the BD BACTEC™ Plus-Aerobic, BacT/Alert(®) SA, and BacT/Alert(®) FA blood culture bottles in 72%, 45.6%, and 23%, respectively, for gram-negative bacteria in 86.6%, 69.2%, and 47.1%, respectively, and for gram-positive bacteria in 60.0%, 28.8%, and 5.4%, respectively. The lack of identification was observed mainly with viridans streptococci. Depending on the blood culture bottles used in routine diagnostic procedures and the protocol for bacterial preparation, the applied MALDI-TOF MS represents an efficient and rapid method for direct bacterial identification.