A real-time analysis of protein transport via the twin arginine translocation pathway in response to different components of the protonmotive force.

The twin arginine translocation (Tat) pathway transports folded protein across the cytoplasmic membrane in bacteria, archaea, and across the thylakoid membrane in plants as well as the inner membrane in some mitochondria. In plant chloroplasts, the Tat pathway utilizes the protonmotive force (PMF) to drive protein translocation. However, in bacteria, it has been shown that Tat transport depends only on the transmembrane electrical potential (Δψ) component of PMF in vitro. To investigate the comprehensive PMF requirement in Escherichia coli, we have developed the first real-time assay to monitor Tat transport utilizing the NanoLuc Binary Technology in E. coli spheroplasts. This luminescence assay allows for continuous monitoring of Tat transport with high-resolution, making it possible to observe subtle changes in transport in response to different treatments. By applying the NanoLuc assay, we report that, under acidic conditions (pH = 6.3), ΔpH, in addition to Δψ, contributes energetically to Tat transport in vivo in E. coli spheroplasts. These results provide novel insight into the mechanism of energy utilization by the Tat pathway.

Evaluation of PhenoMATRIX and PhenoMATRIX PLUS for the screening of MRSA from nasal and inguinal/perineal swabs using chromogenic media.

The objective of this study was to assess the clinical performances of PhenoMATRIX and PhenoMATRIX PLUS for the screening of methicillin-resistant Staphylococcus aureus (MRSA) from nasal and inguinal/perineal ESwabs using chromogenic media. The automated performances were compared to the manual reading. Additionally, we evaluated PhenoMATRIX PLUS for the automatic release of the negative results to the Laboratory Information System (LIS) and the automatic discharge of the negative plates from the incubators. A total of 6,771 non-duplicate specimens were used by PhenoMATRIX as a machine learning model. The validation of these settings was performed on 17,223 non-duplicate specimens. The MRSA positivity rate was 5% (866/17,223). Validated settings were then used by PhenoMATRIX PLUS on another 1,409 non-duplicate specimens. The sensitivities of PhenoMATRIX and PhenoMATRIX PLUS were 99.8% [95% confidence interval (CI), 99.2%-99.9%] and 100% (95% CI, 92.1%-100%), respectively. The specificities of PhenoMATRIX and PhenoMATRIX PLUS were 99.1% (95% CI, 99.0%-99.2%) and 95.2% (95% CI, 93.8%-96.1%), respectively. All the 1,297 MRSA-negative specimens analyzed by PhenoMATRIX PLUS were automatically released and sent to the LIS immediately after availability of the culture image on the WASPLab (100% accuracy). All negative media plates were automatically discarded. PhenoMATRIX PLUS decreases the time spent by technologists on negative plates and ensures optimal usage of the incubators' capacity.

Comparison of three rapid diagnostic tests for bloodstream infections using Benefit-risk Evaluation Framework (BED-FRAME).

Rapid diagnostic tests (RDTs) for bloodstream infections have the potential to reduce time to appropriate antimicrobial therapy and improve patient outcomes. Previously, an in-house, lipid-based, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) method, Fast Lipid Analysis Technique (FLAT MS), has shown promise as a rapid pathogen identification method. In this study, FLAT MS for direct from blood culture identification was evaluated and compared to FDA-cleared identification methods using the Benefit-risk Evaluation Framework (BED-FRAME) analysis. FLAT MS was evaluated and compared to Bruker Sepsityper and bioMérieux BioFire FilmArray BCID2 using results from a previous study. For this study, 301 positive blood cultures were collected from the University of Maryland Medical Center. The RDTs were compared by their sensitivities, time-to-results, hands-on time, and BED-FRAME analysis. The overall sensitivity of all platforms compared to culture results from monomicrobial-positive blood cultures was 88.3%. However, the three RDTs differed in their accuracy for identifying Gram-positive bacteria, Gram-negative bacteria, and yeast. Time-to-results for FLAT MS, Sepsityper, and BioFire BCID2 were all approximately one hour. Hands-on times for FLAT MS, Sepsityper, and BioFire BCID2 were 10 (±1.3), 40 (±2.8), and 5 (±0.25) minutes, respectively. BED-FRAME demonstrated that each RDT had utility at different pathogen prevalence and relative importance. BED-FRAME is a useful tool that can used to determine which RDT is best for a healthcare center.

Development and evaluation of an artificial intelligence for bacterial growth monitoring in clinical bacteriology.

In clinical bacteriology laboratories, reading and processing of sterile plates remain a significant part of the routine workload (30%-40% of the plates). Here, an algorithm was developed for bacterial growth detection starting with any type of specimens and using the most common media in bacteriology. The growth prediction performance of the algorithm for automatic processing of sterile plates was evaluated not only at 18-24 h and 48 h but also at earlier timepoints toward the development of an early growth monitoring system. A total of 3,844 plates inoculated with representative clinical specimens were used. The plates were imaged 15 times, and two different microbiologists read the images randomly and independently, creating 99,944 human ground truths. The algorithm was able, at 48 h, to discriminate growth from no growth with a sensitivity of 99.80% (five false-negative [FN] plates out of 3,844) and a specificity of 91.97%. At 24 h, sensitivity and specificity reached 99.08% and 93.37%, respectively. Interestingly, during human truth reading, growth was reported as early as 4 h, while at 6 h, half of the positive plates were already showing some growth. In this context, automated early growth monitoring in case of normally sterile samples is envisioned to provide added value to the microbiologists, enabling them to prioritize reading and to communicate early detection of bacterial growth to the clinicians.

Automated detection of methicillin-resistant Staphylococcus aureus with the MRSA CHROM imaging application on BD Kiestra Total Lab Automation System.

The virulence of methicillin-resistant Staphylococcus aureus (MRSA) and its potentially fatal outcome necessitate rapid and accurate detection of patients colonized with MRSA in healthcare settings. Using the BD Kiestra Total Lab Automation (TLA) System in conjunction with the MRSA Application (MRSA App), an imaging application that uses artificial intelligence to interpret colorimetric information (mauve-colored colonies) indicative of MRSA pathogen presence on CHROMagar chromogenic media, anterior nares specimens from three sites were evaluated for the presence of mauve-colored colonies. Results obtained with the MRSA App were compared to manual reading of agar plate images by proficient laboratory technologists. Of 1,593 specimens evaluated, 1,545 (96.98%) were concordant between MRSA App and laboratory technologist reading for the detection of MRSA growth [sensitivity 98.15% (95% CI, 96.03, 99.32) and specificity 96.69% (95% CI, 95.55, 97.60)]. This multi-site study is the first evaluation of the MRSA App in conjunction with the BD Kiestra TLA System. Using the MRSA App, our results showed 98.15% sensitivity and 96.69% specificity for the detection of MRSA from anterior nares specimens. The MRSA App, used in conjunction with laboratory automation, provides an opportunity to improve laboratory efficiency by reducing laboratory technologists' labor associated with the review and interpretation of cultures.

Broad-range amplification and sequencing of the rpoB gene: a novel assay for bacterial identification in clinical microbiology.

The rpoB gene has been proposed as a promising phylogenetic marker for bacterial identification, providing theoretically improved species-level resolution compared to the 16S rRNA gene for a range of clinically important taxa. However, its utility in diagnostic microbiology has been limited by the lack of broad-range primers allowing for its amplification from most species with a single PCR assay. Here, we present an assay for broad-range partial amplification and Sanger sequencing of the rpoB gene. To reduce cross-reactivity and allow for rpoB amplification directly from patient samples, primers were based on the dual priming oligonucleotide principle. The resulting amplicon is ~550 base pairs in length and appropriate for species-level identification. Systematic in silico evaluation of a wide selection of taxa demonstrated improved resolution within multiple important genera, including Enterococcus, Fusobacterium, Mycobacterium, Streptococcus, and Staphylococcus species and several genera within the Enterobacteriaceae family. Broad-range rpoB amplification and Sanger sequencing of 115 bacterial isolates provided unambiguous species-level identification for 97 (84%) isolates, as compared to 57 (50%) using a clinical 16S rRNA gene assay. Several unresolved taxonomic matters disguised by the low resolution of the 16S rRNA gene were revealed using the rpoB gene. Using a collection of 33 clinical specimens harboring bacteria and assumed to contain high concentrations of human DNA, the rpoB assay identified the pathogen in 29 specimens (88%). Broad-range rpoB amplification and sequencing provides a promising tool for bacterial identification, improving discrimination between closely related species and making it amenable for use in culture-based and culture-independent diagnostic approaches.

A forced aeration system for microbial culture of multiple shaken vessels suppresses volatilization.

Shake-flask culture, an aerobic submerged culture, has been used in various applications involving cell cultivation. However, it is not designed for forced aeration. Hence, this study aimed to develop a small-scale submerged shaking culture system enabling forced aeration into the medium. A forced aeration control system for multiple vessels allows shaking, suppresses volatilization, and is attachable externally to existing shaking tables. Using a specially developed plug, medium volatilization was reduced to less than 10%, even after 45 h of continuous aeration (~ 60 mL/min of dry air) in a 50 mL working volume. Escherichia coli IFO3301 cultivation with aeration was completed within a shorter period than that without aeration, with a 35% reduction in the time-to-reach maximum bacterial concentration (26.5 g-dry cell/L) and a 1.25-fold increase in maximum concentration. The maximum bacterial concentration achieved with aeration was identical to that obtained using the Erlenmeyer flask, with a 65% reduction in the time required to reach it.

Evaluation of CHROMagar™ B. cepacia agar for the detection of Burkholderia cepacia complex species from sputum samples of patients with cystic fibrosis.

INTRODUCTION: Burkholderia cepacia complex (BCC) are non-fermenting Gram-negative bacteria that can chronically colonize the lungs of people with cystic fibrosis (pwCF), causing a severe and progressive respiratory failure, post-transplant complications and epidemic outbreaks. Therefore, rapid and accurate identification of these bacteria is relevant for pwCF, in order to facilitate early eradication and prevent chronic colonization. However, BCCs are often quite difficult to detect on culture media as they have a slow growth rate and can be hidden by other fast-growing microorganisms, including Pseudomonas aeruginosa and filamentous fungi. MATERIAL AND METHODS: We evaluated the sensitivity of CHROMagar™ B. cepacia agar using 11 isolates from a well-characterized BCC collection, using BCA agar (Oxoid, UK) as a gold standard. We also studied 180 clinical sputum samples to calculate positive (PPV) and negative (NPV) predictive values. Furthermore, we used three of the well-characterized BCC isolates to determine the limit of detection (LOD). RESULTS: Eleven isolates grew on CHROMagar™ B. cepacia at 37ºC after 48 h. The NPV and PPV of CHROMagar™ B. cepacia were 100% and 87.5%, respectively. The LOD of CHROMagar™ B. cepacia was around 1 × 103 CFU/ml, requiring a ten-fold dilution lower bacterial load than BCA for BCC detection. CONCLUSION: CHROMagar™ B. cepacia agar proved to have a very good sensitivity and specificity for the detection of clinical BCCs. Moreover, the chromogenic nature of the medium allowed us to clearly differentiate BCC from other Gram-negative species, filamentous fungi and yeasts, thereby facilitating the identification of contaminants.

Cytomorphological patterns and clinical features of presumptive tubercular lymphadenitis patients and their comparison with bacteriological detection methods: a cross-sectional study.

INTRODUCTION: Tuberculous lymphadenitis (TBLN) is an infection of the lymph node caused by Mycobacterium tuberculosis. Histological diagnoses of presumptive patients are often accompanied by cytomorphological features. However, the sensitivities of these features are often precluded by the variable degrees of narrative similarities compared to other diagnostic modalities. OBJECTIVE: The aim of this study was to investigate and compare the cytomorphological and clinical features of presumptive TBLN patients with bacteriological detection methods. METHODS: A similar cohort of TBLN patients from our previous study who were enrolled prospectively from the ALERT Specialized Hospital, Addis Ababa, Ethiopia, was considered for this analysis. SPSS version 26 was used for data analysis. Descriptive analysis was conducted to characterize the study population using the independent variable and presented with frequency tables. The chi-square test was used to measure the association. A P-value of < 0.05 was considered statistically significant. RESULTS: Using FNAC, 60/126 (47.6%) of the participants were reported to have features consistent with TB. Of the total FNAC-positive cases, many (30/60 and 27/60) showed pattern B (caseous necrosis only) and pattern C (epithelioid granuloma with caseous necrosis), respectively. Strong concordance was observed in Pattern A (abundant caseous necrosis with few epithelioid macrophages) followed by patterns B and C with GeneXpert and MGIT culture (P value < 0.001). Night sweats and alcohol intake were shown to correlate with positive cases as reported by FNAC (P value = 0.008 respectively), GeneXpert (P value = 0.02 & 0.001), and culture methods (P-value = < 0.001 & 0.002). CONCLUSION: Cytomorphological features, particularly patterns A, B, and C, could be considered in the diagnosis of TBLN given their comparable outcomes with bacteriological detection methods. On another note, we recommend that due care and attention be given when treating TBLN patients based solely on clinical presentation, as these diagnostics may be prone to false results, leading to inappropriate administration of anti-TB drugs and other consequences.

Two protocols for the detection of oleaginous bacteria using Oil Red O.

The selection of oleaginous bacteria, potentially applicable to biotechnological approaches, is usually carried out by different expensive and time-consuming techniques. In this study, we used Oil Red O (ORO) as an useful dye for staining of neutral lipids (triacylglycerols and wax esters) on thin-layer chromatography plates. ORO could detect minimal quantities of both compounds (detection limit, 0.0025 mg of tripalmitin or 0.005 mg of cetylpalmitate). In addition, we developed a specific, rapid, and inexpensive screening methodology to detect triacylglycerol-accumulating microorganisms grown on the agar plate. This staining methodology detected 9/13 strains with a triacylglycerol content higher than 20% by cellular dry weight. ORO did not stain polyhydroxyalkanoates-producing bacteria. The four oleaginous strains not detected by this screening methodology exhibited a mucoid morphology of their colonies. Apparently, an extracellular polymeric substance produced by these strains hampered the entry of the lipophilic dye into cells. The utilization of the developed screening methodology would allow selecting of oleaginous bacteria in a simpler and faster way than techniques usually used nowadays, based on unspecific staining protocols and spectrophotometric or chromatographic methods. Furthermore, the use of ORO as a staining reagent would easily characterize the neutral lipids accumulated by microorganisms as reserve compounds. KEY POINTS: • Oil Red O staining is specific for triacylglycerols • Oil Red O staining is useful to detect oleaginous bacteria • Fast and inexpensive staining to isolate oleaginous bacteria from the environment.

Validation of rapid detection methods for Salmonella enterica in green chile.

The objective of this study is to validate the US Food and Drug Administration (FDA) rea-time polymerase chain reaction (qPCR) assay, the Neogen Amplified Nucleic Single Temperature Reaction (ANSR) assay, and the Vitek ImmunoDiagnostic Assay System (VIDAS) SLM procedure against the FDA cultural procedure for Salmonella detection in green chile pepper. Green chile was artificially contaminated with Salmonella according to the FDA guidelines (FDA. Guidelines for the Validation of Microbiological Methods for the FDA Foods Program, 3rd Edition. 2019. www.fda.gov/media/83812/download?attachment (17 March 2024, date last accessed)) at a fractional recovery level (where 50%-25% tests positive and at a level +1 log greater for each organism tested). Enriched samples were tested directly by the ANSR Salmonella test and by qPCR, and were subcultured into Rappaport-Vassiliadis and tetrathionate brilliant green broth for cultural detection and qPCR. For the VIDAS-SLM assay, the selective enrichments were further cultured in M broth before testing. Presumptive salmonellae were confirmed with biochemical tests, serology, and qPCR. All three rapid assays were compared favorably with the FDA-BAM (Bacteriological Analytical Manual) method. No significant differences at P < .05 were found between the procedures using McNemar's χ2 test. The three procedures were found to be rapid and reliable alternatives to cultural detection of Salmonella enterica in green chile.

Edge current and pairing order transition in chiral bacterial vortices.

Bacterial suspensions show turbulence-like spatiotemporal dynamics and vortices moving irregularly inside the suspensions. Understanding these ordered vortices is an ongoing challenge in active matter physics, and their application to the control of autonomous material transport will provide significant development in microfluidics. Despite the extensive studies, one of the key aspects of bacterial propulsion has remained elusive: The motion of bacteria is chiral, i.e., it breaks mirror symmetry. Therefore, the mechanism of control of macroscopic active turbulence by microscopic chirality is still poorly understood. Here, we report the selective stabilization of chiral rotational direction of bacterial vortices in achiral circular microwells sealed by an oil/water interface. The intrinsic chirality of bacterial swimming near the top and bottom interfaces generates chiral collective motions of bacteria at the lateral boundary of the microwell that are opposite in directions. These edge currents grow stronger as bacterial density increases, and, within different top and bottom interfaces, their competition leads to a global rotation of the bacterial suspension in a favored direction, breaking the mirror symmetry of the system. We further demonstrate that chiral edge current favors corotational configurations of interacting vortices, enhancing their ordering. The intrinsic chirality of bacteria is a key feature of the pairing order transition from active turbulence, and the geometric rule of pairing order transition may shed light on the strategy for designing chiral active matter.

Evaluation of a Novel Benchtop Tool for Acceleration of Sample Preparation for MALDI-TOF Mass Spectrometry.

During the past decade, MALDI-TOF mass spectrometry (MS) has become a standard method for identification of bacteria and yeasts. Nonetheless, further optimization of the identification process is important to streamline workflows and save resources. This study evaluated the application of a multipurpose benchtop tool, MBT FAST Shuttle IVD, for accelerated drying of liquid assay components (matrix, formic acid, and/or sample) on a MALDI target. A total of 50 bacterial and fungal isolates were subjected to three different sample preparation procedures prior to the identification by MALDI-TOF MS: direct transfer (DT), extended direct transfer (eDT), and protein extraction (PE). Compared to conventional drying at room temperature, the preparation was performed with standardized heating of the MALDI target on the MBT FAST Shuttle. During DT, eDT, and PE, 56.7% (P < 0.001), 56.8% (P < 0.001), and 52.8% (P < 0.001) of time for matrix drying were saved by using the MBT FAST Shuttle, respectively. Applying the MBT FAST Shuttle, 57.5% (P < 0.001) of time for drying of formic acid were saved for eDT and 57.5% (P < 0.001) of time for sample drying were saved for PE. A significant improvement of the identification rates and scores was observed with MBT FAST Shuttle for eDT (P = 0.001) and PE (P = 0.008) methods, while the effect on identification quality for DT was not statistically significant (P = 0.16). In conclusion, the use of the MBT FAST Shuttle shortened the drying time of assay components by about a half for all preparation methods. Moreover, positive effect on identification success was observed.

Evaluation of a new rapid immunochromatographic assay for the detection of GES-producing Gram-negative bacteria.

BACKGROUND: As carbapenemase-producing Enterobacterales are increasingly reported worldwide, their rapid detection is crucial to reduce their spread and prevent infections and outbreaks. Lateral flow immunoassays (LFIAs) have become major tools for the detection of carbapenemases. However, as for most commercially available assays, only the five main carbapenemases are targeted. OBJECTIVES: Here, we have developed and evaluated an LFIA prototype for the rapid and reliable detection of the increasingly identified GES-type ß-lactamases. METHODS: The GES LFIA was validated on 103 well-characterized Gram-negative isolates expressing various ß-lactamases grown on Mueller-Hinton (MH) agar, chromogenic, and chromogenic/selective media. RESULTS: The limit of detection of the assay was 106 cfu per test with bacteria grown on MH agar plates. GES LFIA accurately detected GES-type ß-lactamases irrespective of the culture media and the bacterial host. The GES LFIA was not able to distinguish between GES-ESBLs and GES-carbapenemases. Because GES enzymes are still rare, their detection as an ESBL or a carbapenemase remains important, especially because extensive use of carbapenems to treat ESBL infections may select for GES variants capable of hydrolysing carbapenems. CONCLUSIONS: The GES LFIA is efficient, rapid and easy to implement in the routine workflow of a clinical microbiology laboratory for the confirmation of GES-type ß-lactamases. Combining it with immunochromatographic assays targeting the five main carbapenemases (KPC, NDM, VIM, IMP and OXA-48) would improve the overall sensitivity for the most frequently encountered carbapenemases and ESBLs, especially in non-fermenters.

Proof of concept for multiplex detection of antibodies against Chlamydia species in chicken serum using a bead-based suspension array with peptides as antigens.

The available differentiating tests for Chlamydia are based on detection of genetic material and only give information about the actual infection status, but reveal nothing of past infections. As the use of serological methods increases the window of detection, the goal of this study was to investigate if it is possible to develop a differentiating serological test for antibodies against Chlamydia species in chicken sera. Focus was on C. psittaci, C. gallinacea, and two closely related species, i.e. C. abortus and C. avium. To enable differentiating serology, a bead-based Luminex suspension array was constructed, using peptides as antigens, derived from known immunoreactive Chlamydia proteins. For the majority of these peptides, species-specific seroreactivity in mammalian sera has been reported in literature. The suspension array correctly identified antibodies against various Chlamydia species in sera from experimentally infected mice, and was also able to differentiate between antibodies against C. psittaci and C. gallinacea in sera from experimentally infected chickens. In field sera, signals were difficult to interpret as insufficient sera from experimentally infected chickens were available for evaluating the seroreactivity of all peptides. Nevertheless, results of the suspension array with field sera are supported by published data on the occurrence of C. gallinacea in Dutch layers, thereby demonstrating the proof of concept of multiplex serology for Chlamydial species in poultry.

Impact of randomized blinded rechecking program on the performance of the AFB Microscopy Laboratory Network in Uganda: a decadal retrospective study.

BACKGROUND: Smear microscopy has remained the initial diagnostic test for presumptive tuberculosis (TB) patients in health facilities without the World Health Organization (WHO) recommended rapid diagnostic tools. In the Uganda TB laboratory network, the technique remains the only tool to monitor response to treatment among drug susceptible TB patients, with the country currently having over 1,600 microscopy TB testing units. It has been evidenced that acid-fast bacilli (AFB) microscopy's yield highly depends on the staining technique and reading ability of the laboratory personnel. For the quality of TB testing in the country, the TB control program set up a Randomized Blinded Rechecking (RBRC) program in 2008 to monitor the testing performance of laboratories to continuously improve the reliability and efficiency of results. This is the first study to determine the effectiveness and impact of the RBRC program on the performance of the participating laboratories in Uganda. METHODS: This was a retrospective cross-sectional study based on a record review of the RBRC's annual results compilations between January 2008 and December 2017. RESULTS: Between January 2008 and December 2017, a total of 265,523 smears were re-checked during the RBRC program. The number of enrolled laboratories in the RBRC program rose from 660 to 2008 to 1,406 in 2017. The RBRC program resulted in a statistically significant reduction in microscopy errors, with false positives decreasing from 12.8% to 2008 to 7.6% in 2017, false positive errors decreasing from 10 to 6.3%, false negative errors decreasing from 2.9 to 0.7%, quantification errors decreasing from 6.0 to 1.8%, and the overall sensitivity of smear microscopy compared to the controllers increased with statistical significance from 93 to 97%. CONCLUSION: The study reveals an overall significant error reduction and an improved sensitivity of smear microscopy upon continuous implementation of the RBRC program in an AFB microscopy TB laboratory network. Implementation of a RBRC program is crucial and essential to maintaining a reliable TB laboratory service that can facilitate accurate diagnosis and offset the disadvantages of using smear microscopy.

SERS-based rapid susceptibility testing of commonly administered antibiotics on clinically important bacteria species directly from blood culture of bacteremia patients.

Bloodstream infections are a growing public health concern due to emerging pathogens and increasing antimicrobial resistance. Rapid antibiotic susceptibility testing (AST) is urgently needed for timely and optimized choice of antibiotics, but current methods require days to obtain results. Here, we present a general AST protocol based on surface-enhanced Raman scattering (SERS-AST) for bacteremia caused by eight clinically relevant Gram-positive and Gram-negative pathogens treated with seven commonly administered antibiotics. Our results show that the SERS-AST protocol achieves a high level of agreement (96% for Gram-positive and 97% for Gram-negative bacteria) with the widely deployed VITEK 2 diagnostic system. The protocol requires only five hours to complete per blood-culture sample, making it a rapid and effective alternative to conventional methods. Our findings provide a solid foundation for the SERS-AST protocol as a promising approach to optimize the choice of antibiotics for specific bacteremia patients. This novel protocol has the potential to improve patient outcomes and reduce the spread of antibiotic resistance.

Multiple Cross Displacement Amplification Coupled with Lateral Flow Biosensor (MCDA-LFB) for rapid detection of Legionella pneumophila.

BACKGROUND: Legionella pneumophila is an opportunistic waterborne pathogen of significant public health problems, which can cause serious human respiratory diseases (Legionnaires' disease). Multiple cross displacement amplification (MCDA), a isothermal nucleic acid amplification technique, has been applied in the rapid detection of several bacterial agents. In this report, we developed a MCDA coupled with Nanoparticles-based Lateral Flow Biosensor (MCDA-LFB) for the rapid detection of L. pneumophila. RESULTS: A set of 10 primers based on the L. pneumophila specific mip gene to specifically identify 10 different target sequence regions of L. pneumophila was designed. The optimal time and temperature for amplification are 57 min and 65 °C. The limit of detection (LoD) is 10 fg in pure cultures of L. pneumophila. No cross-reaction was obtained and the specificity of MCDA-LFB assay was 100%. The whole process of the assay, including 20 min of DNA preparation, 35 min of L. pneumophila-MCDA reaction, and 2 min of sensor strip reaction, took a total of 57 min (less than 1 h). Among 88 specimens for clinical evaluation, 5 (5.68%) samples were L. pneumophila-positive by MCDA-LFB and traditional culture method, while 4(4.55%) samples were L. pneumophila-positive by PCR method targeting mip gene. Compared with culture method, the diagnostic accuracy of MCDA-LFB method was higher. CONCLUSIONS: In summary, the L. pneumophila-MCDA-LFB method we successfully developed is a simple, fast, reliable and sensitive diagnostic tool, which can be widely used in basic and clinical laboratories.

Bacterial Cell Division: Nonmodels Poised to Take the Spotlight.

The last three decades have witnessed an explosion of discoveries about the mechanistic details of binary fission in model bacteria such as Escherichia coli, Bacillus subtilis, and Caulobacter crescentus. This was made possible not only by advances in microscopy that helped answer questions about cell biology but also by clever genetic manipulations that directly and easily tested specific hypotheses. More recently, research using understudied organisms, or nonmodel systems, has revealed several alternate mechanistic strategies that bacteria use to divide and propagate. In this review, we highlight new findings and compare these strategies to cell division mechanisms elucidated in model organisms.

Present and Future of Culturing Bacteria.

The cultivation of bacteria is highly biased toward a few phylogenetic groups. Many of the currently underexplored bacterial lineages likely have novel biosynthetic pathways and unknown biochemical features. New cultivation concepts have been developed based on an improved understanding of the ecology of previously not-cultured bacteria. Particularly successful were improved media that mimic the natural types and concentrations of substrates and nutrients, high-throughput cultivation techniques, and approaches that exploit biofilm formation and bacterial interactions. Metagenomics and single-cell genomics can reveal unknown metabolic features of not-yet-cultured bacteria and, if complemented by culture-independent physiological analyses, will help to target functional novelty more efficiently. However, numerous novel types of bacteria that were initially enriched subsequently escaped isolation. Future cultivation work will therefore need to focus on improved subcultivation, purification, and preservation techniques to recover and utilize a larger fraction of microbial diversity.