Name: identification of rare bacterial pathogens by 16s rrna gene sequencing and maldi-tof ms
Uploaded: 2016-07-11T13:00:00
Description: Watch this Scientific Journal Video about Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS at JoVE.com

The authors would like to thank Prof. Enno Jacobs for his continuing support.

1. Extraction of Bacterial DNA
<ol>
	<li>Preparation of PBS Solution
	<ol>
		<li>Weigh 1.65 g Na2HPO4 x 2H2O, 0.22 g NaH2PO4 x 2H2O and 8.80 g NaCl in a flask and fill with distilled water to a final volume of 1,000 ml. Adjust the pH to 7.4. For final use filter the solution through a bacteria-proof (0.22 &#181;m) filter.</li>
	</ol>
	</li>
	<li>DNA Extraction of Gram-negative Bacteria
	<ol>
		<li>Streak the patient material on appropriate culture media ...

Both MALDI-TOF MS and 16S rRNA gene sequencing offer the possibility to identify large numbers of different bacteria. MALDI-TOF MS is a fast and inexpensive method, which is easy to handle and large databases of bacterial mass spectra are available. For this reason, MALDI-TOF MS is a rapid, cost effective and reliable method to conduct screening studies focused on rare bacterial pathogens 17,20,39,51. In a prospective study comparing MALDI-TOF MS with other phenotypic identification methods, Seng et al.</e...

Secure identification of rare pathogens in routine diagnostics is hampered by the fact that classical cultural and biochemical methods are cumbersome and sometimes questionable. Furthermore, a diagnostic microbiology laboratory has to process a large number of pathogens, ranging from a few hundred to several thousands, daily, which requires the use of automated systems. In addition to the management of a high daily throughput, the precise identification of bacterial species is needed. This is warranted since they differ in their antimicrobial susceptibility pattern and therefore correct identification provides the clinician with essential information to choose appropriate antibiotics (e.g., Enterococcus spp., Acinetobacter spp.) 12,43.
Automated microbial identification systems (aMIS) apply standardized sets of enzymatic reactions to characterize the metabolic properties of bacterial isolates 13,15,16,26,27. Although the cartridges used in these systems utilize a large number of different biochemical reactions, e.g., 47 in the GN card of the aMIS used in this study 52, this strategy permits secure identification only for a limited set of bacteria. Furthermore, the database, an advanced expert system, is clearly focused on detection of relevant and highly relevant bacteria of medical importance 13,15,16,36. Two further systems, widely used in laboratories, also apply this biochemical approach for bacterial identification. Recent studies demonstrate a comparable identification accuracy between the aMIS used in this study and one of the competitors (93.7% and 93.0% respectively), while the 3rd aMIS has an identification accuracy of only 82.4% on species level 35. Such discrepancies may be explained by the quality of the underlying identification data references, the versions of kits and software, differences in metabolism and proficiency of technical personnel 35,36.
Two automated MALDI-TOF MS systems (MALDI-TOF microbial identification system, mMIS) are mainly used. These systems allow for detection of a large number of bacterial species based on their protein fingerprint mass spectra. For instance, the database of the mMIS used contains 6,000 reference spectra. Identification systems based on mass spectrometry offer fast and reliable detection of a great variety of microorganisms including rare pathogens 11,48,51. To date only a few direct comparisons are available between the mMIS used in this study and its competitor 19,33. According to Daek et al. both systems provide a similar high rate of identification accuracy, but the mMIS used in this study seems to be more reliable in species identification 19.
Similarly, molecular techniques addressing well conserved but also distinct genes (e.g., 16S rDNA or rpoB) permit a clear species identification 3,22,61. Among these, the 16S rDNA is the most widely used housekeeping gene because of its presence in all bacteria 34. Its function remains unchanged and finally, with roughly 1,500 bp, it is long enough to be suitable for bio-informatics 14,34. Many researchers regard 16S rRNA gene analysis as the &#34;gold-standard&#34; for bacterial identification 21. This is due to the fact that few laboratories use DNA-DNA hybridization techniques to date for identification of rare or new bacteria 14,34. Additionally, more and more databases are available which can be used for 16S rRNA gene analysis 50. However, it has to be taken into account that 16S rDNA based detection systems have a limited sensitivity compared to standard PCR protocols. Moreover, the molecular approach is sophisticated, time consuming and requires highly trained personnel as well as dedicated laboratory facilities and is, therefore, not easily implemented into routine diagnostics 55. Furthermore, it has been shown that the combination of at least two different methods of bacterial identification leads to highly accurate strain identification. The combination of MALDI-TOF MS and 16S rDNA sequencing permits the identification of large numbers of different bacterial species with high accuracy. Recently the combination of MALDI-TOF MS and 16S rRNA gene analysis was presented for bacterial identification studying epidemiological questions and rare pathogens 56.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CHROMASOLV, HPLC grade water, 1 L</td>
			<td>Sigma-Aldrich Chemie, M&#252;nchen, Germany</td>
			<td>270733</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Lyser LT</td>
			<td>Qiagen, Hilden, Germany</td>
			<td>85600</td>
			<td>Oscillating Homogenizer</td>
		</tr>
		<tr>
			<td>Glass-beads 1,0mm</td>
			<td>VWR International, Darmstadt, Germany</td>
			<td>412-2917</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermomixer 5436</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td>2050-100-05</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAamp DNA Mini Kit (250)</td>
			<td>Qiagen, Hilden, Germany</td>
			<td>51306</td>
			<td></td>
		</tr>
		<tr>
			<td>Taq PCR Core Kit (1000 U)</td>
			<td>Qiagen, Hilden, Germany</td>
			<td>201225</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">Forward Primer TPU1 (5&#180;-AGA GTT TGA TCM TGG CTC AG-3&#8217;)</td>
			<td rowspan="2">biomers.net GmbH, Ulm, Germany&#160;</td>
			<td rowspan="2">-</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">Reverse Primer RTU4 (5&#180;-TAC CAG GGT ATC TAA TCC TGT T-3&#180;)</td>
			<td rowspan="2">biomers.net GmbH, Ulm, Germany&#160;</td>
			<td rowspan="2">-</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
		</tr>
		<tr>
			<td>Mastercycler&#160;</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td>-&#160;</td>
			<td>Thermocylcer</td>
		</tr>
		<tr>
			<td>Reaction tube 1.5 mL</td>
			<td>SARSTEDT, N&#252;mbrecht, Germany</td>
			<td>72,692</td>
			<td></td>
		</tr>
		<tr>
			<td>Reaction tube 2 mL</td>
			<td>SARSTEDT, N&#252;mbrecht, Germany</td>
			<td>72,693,005</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR 8er-CapStrips</td>
			<td>Biozym Scientific, Hessisch Oldendorf, Germany</td>
			<td>711040X</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR 8er-SoftStrips</td>
			<td>Biozym Scientific, Hessisch Oldendorf, Germany</td>
			<td>711030X</td>
			<td></td>
		</tr>
		<tr>
			<td>Sharp R-ZV11&#160;</td>
			<td>Sharp Electronics, Hamburg, Germany</td>
			<td>-</td>
			<td>Microwave</td>
		</tr>
		<tr>
			<td>Titriplex III (EDTA Na2-salt dehydrate; 1 kg)</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>1084211000</td>
			<td></td>
		</tr>
		<tr>
			<td>SeaKem LE Agarose</td>
			<td rowspan="2">Biozym Scientific, Hessisch Oldendorf, Germany</td>
			<td rowspan="2">849006</td>
			<td></td>
		</tr>
		<tr>
			<td>(2 x 500 g)</td>
			<td></td>
		</tr>
		<tr>
			<td>SmartLadder SF - 100 to 1000 bp</td>
			<td>Eurogentec, L&#252;ttich, Belgium</td>
			<td>MW-1800-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromphenol blue (25 g)</td>
			<td>Sigma-Aldrich Chemie, M&#252;nchen, Germany</td>
			<td>B0126</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene cyanol FF (10 g)</td>
			<td>Sigma-Aldrich Chemie, M&#252;nchen, Germany</td>
			<td>X4126</td>
			<td></td>
		</tr>
		<tr>
			<td>ComPhor L Maxi&#160;</td>
			<td>Biozym, Hessisch Oldendorf, Germany</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethidium bromide solution 1&#160;%(10 mL)</td>
			<td>Carl Roth, Karlsruhe, Germany</td>
			<td>2218.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Doc 2000</td>
			<td>Bio-Rad Laboratories, M&#252;nchen, Germany</td>
			<td>-</td>
			<td>Gel-documentation system&#160;</td>
		</tr>
		<tr>
			<td>ExoSAP-IT (500 reactions)</td>
			<td>Affymetrix UK, Wooburn Green, High Wycombe, United Kingdom</td>
			<td>78201</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffer (10 x) with EDTA&#160;</td>
			<td>Life Technologies, Darmstadt, Germany</td>
			<td>402824</td>
			<td></td>
		</tr>
		<tr>
			<td>BigDye Terminator Kit v1.1</td>
			<td>Life Technologies, Darmstadt, Germany</td>
			<td>4337450</td>
			<td></td>
		</tr>
		<tr>
			<td>Hi-Di formamide (25 mL)</td>
			<td>Life Technologies, Darmstadt, Germany</td>
			<td>4311320</td>
			<td></td>
		</tr>
		<tr>
			<td>DyeEx 2.0 Spin Kit (250)</td>
			<td>Qiagen, Hilden, Germany</td>
			<td>63206</td>
			<td></td>
		</tr>
		<tr>
			<td>3130 Genetic Analyzer</td>
			<td>Life Technologies, Darmstadt, Germany</td>
			<td>-</td>
			<td>Sequenzer</td>
		</tr>
		<tr>
			<td>MicroAmp optical 96-well reaction plate with barcode</td>
			<td>Life Technologies, Darmstadt, Germany</td>
			<td>4306737</td>
			<td></td>
		</tr>
		<tr>
			<td>3130 Genetic Analyzer, plate base 96-well</td>
			<td>Life Technologies, Darmstadt, Germany</td>
			<td>4317237</td>
			<td></td>
		</tr>
		<tr>
			<td>3130 Genetic Analyzer, plate retainer 96-well</td>
			<td>Life Technologies, Darmstadt, Germany</td>
			<td>4317241</td>
			<td></td>
		</tr>
		<tr>
			<td>3130 Genetic Analyzer, well plate septa</td>
			<td>Life Technologies, Darmstadt, Germany</td>
			<td>4315933</td>
			<td></td>
		</tr>
		<tr>
			<td>3130 Genetic Analyzer, POP-7 Polymer, 7 mL</td>
			<td>Life Technologies, Darmstadt, Germany</td>
			<td>4352759</td>
			<td></td>
		</tr>
		<tr>
			<td>3130 Genetic Analyzer, 4-Capillary Array, 50 cm</td>
			<td>Life Technologies, Darmstadt, Germany</td>
			<td>4333466</td>
			<td></td>
		</tr>
		<tr>
			<td>Sequencing Analysis Software 5.4</td>
			<td>Life Technologies, Darmstadt, Germany</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>microflex (the MALDI TOF MS maschine)</td>
			<td>Bruker Daltonik, Bremen, Germany</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>MALDI Biotyper (the MALDI TOF MS system)</td>
			<td>Bruker Daltonik, Bremen, Germany</td>
			<td>-</td>
			<td>our mMIS</td>
		</tr>
		<tr>
			<td>VITEK MS&#160;</td>
			<td>bioM&#233;rieux, N&#252;rtingen, Germany&#160;</td>
			<td></td>
			<td>2nd mMis&#160;</td>
		</tr>
		<tr>
			<td>flexControl 3.4 (control software)</td>
			<td>Bruker Daltonik, Bremen, Germany</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotyper Realtime Classification 3.1 (RTC), (analysis software)</td>
			<td>Bruker Daltonik, Bremen, Germany</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-cyano-4-hydroxycinnamic acid, HCCA, 1&#160;g</td>
			<td>Bruker Daltonik, Bremen, Germany</td>
			<td>201344</td>
			<td></td>
		</tr>
		<tr>
			<td>Peptide Calibration Standard II</td>
			<td>Bruker Daltonik, Bremen, Germany</td>
			<td>222570</td>
			<td></td>
		</tr>
		<tr>
			<td>MSP 96 target polished steel</td>
			<td>Bruker Daltonik, Bremen, Germany</td>
			<td>8224989</td>
			<td></td>
		</tr>
		<tr>
			<td>peqgreen&#160;</td>
			<td>peqlab&#160;</td>
			<td>37-5010</td>
			<td></td>
		</tr>
		<tr>
			<td>MALDI Biotyper Galaxy&#160;</td>
			<td>Bruker Daltonik, Bremen, Germany</td>
			<td>Part No. 1836007&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Vitek 2&#160;</td>
			<td>bioM&#233;rieux, N&#252;rtingen, Germany&#160;</td>
			<td></td>
			<td>our aMis&#160;</td>
		</tr>
		<tr>
			<td>MicroScan&#160;</td>
			<td>Beckman Coulter&#160;</td>
			<td></td>
			<td>2nd aMis&#160;</td>
		</tr>
		<tr>
			<td>BD Phoenix&#8482; Automated Microbiology System</td>
			<td>BD</td>
			<td></td>
			<td>3rd aMis&#160;</td>
		</tr>
		<tr>
			<td>Staphylococcus aureus&#160;subsp.&#160;aureus&#160;Rosenbach (ATCC&#174;&#160;25923&#8482;)</td>
			<td>ATCC&#160;</td>
			<td></td>
			<td>postive control for PCR&#160;</td>
		</tr>
	</tbody>
</table>

identification of rare bacterial pathogens by 16s rrna gene sequencing and maldi-tof ms

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in immunocompromised patients. In most cases only few data, mostly published as case reports, are available which investigate the role of such pathogens as an infectious agent. Therefore, in order to clarify the pathogenic character of such microorganisms, it is necessary to conduct epidemiologic studies which include large numbers of these bacteria. The methods used in such a surveillance study have to meet the following criteria: the identification of the strains has to be accurate according to the valid nomenclature, they should be easy to handle (robustness), economical in routine diagnostics and they have to generate comparable results among different laboratories. Generally, there are three strategies for identifying bacterial strains in a routine setting: 1) phenotypic identification characterizing the biochemical and metabolic properties of the bacteria, 2) molecular techniques such as 16S rRNA gene sequencing and 3) mass spectrometry as a novel proteome based approach. Since mass spectrometry and molecular approaches are the most promising tools for identifying a large variety of bacterial species, these two methods are described. Advances, limitations and potential problems when using these techniques are discussed.

MALDI-TOF MS is a novel, fast and inexpensive method for microbiological routine diagnostics. Bacterial species identification by MALDI-TOF MS produces spectra mainly composed of ribosomal proteins but also other &#34;very conserved proteins with house-keeping functions affected to a minimal extent by environmental conditions&#34; 17.The database of this mMIS contains a large set of reference spectra and even bacteria which are rarely found in clinical isolates can be securely ...

Watch this Scientific Journal Video about Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS at JoVE.com

Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and molecular techniques (16S rRNA gene sequencing) permit the identification of rare bacterial pathogens in routine diagnostics. The goal of this protocol lies in the combination of both techniques which leads to more accurate and reliable data.

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in ...

The overall goal of this MALDI-TOF Mass Spectrometry protocol is to improve typing of rare pathogens through a rapid and cost-effective procedure. This method can help to answer key questions in the field of diagnostic microbiology, it allows rapid edification of rare bacterial pathogens. Today, we want to talk about odoratimimus and bacteria of the genus Myroides.To demonstrate the method and to talk about the interpretation of the results. Generally, individuals new to this method might find it difficult since transfer of bacteria, overlay with metrics, and interpretation of results require technical skills and microbiological expertise. We first had the idea to use MALD-TOF MS to screen for rare bacteria when we had a patient in our hospital suffering from a diabetic food infection caused by myroides odoratimus.Begin this protocol with the extraction of bacterial DNA, 16S ribosomal DNA PCR and Sanger sequencing as described in the text protocol. Use a wooden toothpick to transfer a single bacterial colony to a well of a 96 well steel target. Spot one microliter of 70%formic acid on top of the air-dried organism on the steel target, and leave to dry for approximately two to three minutes.Overlay the spot with one microliter of matrix solution containing 50 mg/ml of CHCA in an organic solvent composed of 50%acetonitrile, 2.5 trifluoroacetic acid, and 47.5%water. Let the smear and overlaid matrix air dry for about five minutes in a hood. To introduce the samples, aerate the sample loading port of the mass spectrometer by pressing the in/out button of the instrument.Open the loading port and insert the MALDI-TOF MS target plate with the dried bacterial smear plus matrix. Close the port and evacuate again. Observe the vacuum and when it reaches 4.5 million millibar start the measurement.Open the MMIS analysis software and click the file menu. Select New Classification"and a new window called MALDI Biotyper Real Time Classification Wizard"should open. Type the project name in the field Project Name"and click New, under the new dialogue box named New Project, check the project name and proceed by clicking the Okay"button.In the MALDI Biotyper Real Time Classification Wizard, observe the Analyte placement window. Select target positions, right click any selected position, and select Add samples. In the automatically opened table view, type in the sample name, sample ID, and optionally, a comment.Click the Next"button to proceed. Next, observe the selection of MALDI Biotyper methods window. Select Bruker Taxonomy"from the MSPs from Taxonomy Trees and click Next"to continue.Then, observe the project summary window. Check the inserted entries given above for the actual classification project. Start the classification run by clicking the Finish"button.Measurements start automatically when the appropriate vacuum is reached. Follow the classification run until the measurement is successfully completed. Once the measurement is complete, open the menu View"and click Results"to view the results table in the MALDI Biotyper Real Time Classification Project One.View the Bruker-Daltonik MALDI Biotyper classification results as an html file in the default web browser. Print and save the results. Shown here are spectra obtained by MALDI-TOF MS that are measured and classified in real time.The spectra are assigned a species name and a score value reflecting the quality of the measurement. Scores above 2.300 represent a highly-probable species identification. A score between 2.000 and 2.300 indicates a secure species identification.A score below 1.700 and 2.000 designates a probable species identification. And a score below 1.700 is non-reliable. The identification of bacterial pathogens grown on a pure culture on a solid medium takes two to three minutes.It is important to remember that rare bacteria pathogens can only be found with MALDI-TOF MS when the instruments reliably contains the appropriate spectra. Otherwise, it has to be expunged by integrating MaSDA spectra from typed strains of their characterized isolates. In the case where score results do not lead to an unequivocal strain identification, further methods, such as biochemical profiling or 16S rDNA sequencing can be applied.Though this method can provide insight into bacterial detection, it can also be applied to other microorganisms such as fungi or anaerobes. Furthermore, bacteria-resistance can be measured. Don't forget working with a matrix which is a hazardous substance, wear gloves, protective goggles, and work under a hood.Furthermore, it is absolutely necessary to dry the matrix and smear thoroughly to avoid splash overs in the analyzation chamber.

identification-rare-bacterial-pathogens-16s-rrna-gene-sequencing

Research

JoVE Journal

Immunology and Infection

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. Although the natural occurrence of both diseases&#39; is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease&#39;s inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management.
Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens 2-4. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant &#34;light-tagged&#34; reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage 5-8. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens 7.
For demonstration purposes, here we describe the method for the phage-mediated detection of a known Y. pestis isolate using a luxAB reporter phage constructed from the CDC plague diagnostic phage &#934;A1122 6,7 (Figure 1). A similar method, with minor modifications (e.g. change in growth temperature and media), may be used for the detection of B. anthracis isolates using the B. anthracis reporter phage W&#946;::luxAB 8. The method describes the phage-mediated transduction of a biolumescent phenotype to cultivated Y. pestis cells which are subsequently measured using a microplate luminometer. The major advantages of this method over the traditional phage lysis assays is the ease of use, the rapid results, and the ability to test multiple samples simultaneously in a 96-well microtiter plate format.
<img alt="Figure 1" src="/files/ftp_upload/2740/2740fig1.jpg" /> 
Figure 1. Detection schematic. The phage are mixed with the sample, the phage infects the cell, luxAB are expressed, and the cell bioluminesces. Sample processing is not necessary; the phage and cells are mixed and subsequently measured for light.

&#039;Bioluminescent&#039; Reporter Phage for the Detection of Category A Bacterial Pathogens

A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis.

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen interactions 1. The major cell types of the innate immune system, macrophages and neutrophils, develop during the first days of embryogenesis prior to the maturation of lymphocytes that are required for adaptive immune responses. The ease of obtaining large numbers of embryos, their accessibility due to external development, the optical transparency of embryonic and larval stages, a wide range of genetic tools, extensive mutant resources and collections of transgenic reporter lines, all add to the versatility of the zebrafish model. Salmonella enterica serovar Typhimurium (S. typhimurium) and Mycobacterium marinum can reside intracellularly in macrophages and are frequently used to study host-pathogen interactions in zebrafish embryos. The infection processes of these two bacterial pathogens are interesting to compare because S. typhimurium infection is acute and lethal within one day, whereas M. marinum infection is chronic and can be imaged up to the larval stage 2, 3. The site of micro-injection of bacteria into the embryo (Figure 1) determines whether the infection will rapidly become systemic or will initially remain localized. A rapid systemic infection can be established by micro-injecting bacteria directly into the blood circulation via the caudal vein at the posterior blood island or via the Duct of Cuvier, a wide circulation channel on the yolk sac connecting the heart to the trunk vasculature. At 1 dpf, when embryos at this stage have phagocytically active macrophages but neutrophils have not yet matured, injecting into the blood island is preferred. For injections at 2-3 dpf, when embryos also have developed functional (myeloperoxidase-producing) neutrophils, the Duct of Cuvier is preferred as the injection site. To study directed migration of myeloid cells towards local infections, bacteria can be injected into the tail muscle, otic vesicle, or hindbrain ventricle 4-6. In addition, the notochord, a structure that appears to be normally inaccessible to myeloid cells, is highly susceptible to local infection 7. A useful alternative for high-throughput applications is the injection of bacteria into the yolk of embryos within the first hours after fertilization 8. Combining fluorescent bacteria and transgenic zebrafish lines with fluorescent macrophages or neutrophils creates ideal circumstances for multi-color imaging of host-pathogen interactions. This video article will describe detailed protocols for intravenous and local infection of zebrafish embryos with S. typhimurium or M. marinum bacteria and for subsequent fluorescence imaging of the interaction with cells of the innate immune system.

Transparent zebrafish embryos have proved useful model hosts to visualize and functionally study interactions between innate immune cells and intracellular bacterial pathogens, such as Salmonella typhimurium and Mycobacterium marinum. Micro-injection of bacteria and multi-color fluorescence imaging are essential techniques involved in the application of zebrafish embryo infection models.

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen ...

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri or Mycobacterium tuberculosis. The approach makes use of CCF4, a FRET reporter sensitive to &beta;-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, &beta;-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri as well as multiple mycobacterial strains such as Mycobacterium marinum, Mycobacterium bovis, and Mycobacterium tuberculosis.

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

Whole-cell MALDI-TOF Mass Spectrometry is an Accurate and Rapid Method to Analyze Different Modes of Macrophage Activation

MALDI-TOF is an extensively used mass spectrometry technique in chemistry and biochemistry. It has been also applied in medicine to identify molecules and biomarkers. Recently, it has been used in microbiology for the routine identification of bacteria grown from clinical samples, without preparation or fractionation steps. We and others have applied this whole-cell MALDI-TOF mass spectrometry technique successfully to eukaryotic cells. Current applications range from cell type identification to quality control assessment of cell culture and diagnostic applications. Here, we describe its use to explore the various polarization phenotypes of macrophages in response to cytokines or heat-killed bacteria. It allowed the identification of macrophage-specific fingerprints that are representative of the diversity of proteomic responses of macrophages. This application illustrates the accuracy and simplicity of the method. The protocol we described here may be useful for studying the immune host response in pathological conditions or may be extended to wider diagnostic applications.

This protocol describes the use of whole-cell MALDI-TOF mass spectrometry on eukaryotic cells. Here, we illustrate the accuracy of this technique by analyzing the multiple activation states of macrophages in response to their microenvironment.

MALDI-TOF is an extensively used mass spectrometry technique in chemistry and biochemistry. It has been also applied in medicine to identify molecules and ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal meningitis worldwide resulting in more than 625,000 deaths per year worldwide.&#160;Although electroporation has been developed for the transformation of plasmids in Cryptococcus, only&#160;biolistic&#160;delivery provides&#160;an effective means to transform&#160;linear&#160;DNA&#160;that can be integrated into the genome by homologous recombination.&#160;
Acetate has been shown to be a major fermentation product during cryptococcal infection, but the significance of this is not yet known.&#160;A bacterial pathway composed of the enzymes xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp) and acetate kinase (Ack) is one of three potential pathways for acetate production in C. neoformans. Here, we demonstrate the biolistic transformation of a construct, which has the gene encoding Ack fused to the fluorescent tag mCherry, into&#160;C. neoformans. We then confirm integration of the ACK-mCherry fusion into the&#160;ACK&#160;locus.

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

Biolistic transformation is a method used to generate stable integration of DNA into the genome of the opportunistic pathogen Cryptococcus neoformans through homologous recombination. We will demonstrate biolistic transformation of a construct, which has the gene encoding acetate kinase fused to the fluorescent tag mCherry into&#160;C. neoformans.

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal ...

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been reported using various methods, including electron microscopy, immunohistochemistry or immunofluorescence using bacteria-specific antibodies, and fluorescent in situ hybridization (FISH) using a fluorescence-labeled oligonucleotide probe. Nevertheless, these methods are not widely used due to technical limitation or difficulties. Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled DNA probes has been introduced. The paraffin-embedded tissues are the most common form of biopsy tissues available from pathology banks. Bacteria can be detected either in a species-specific or universal manner. Bacterial signals are detected as either discrete forms (coccus, rod, fusiform, and hairy form) of bacteria or dispersed forms. The technique allows other histological information to be obtained: the epithelia, connective tissue, inflammatory infiltrates, and blood vessels are well distinguished. This method can be used to study the role of bacteria in various diseases, such as periodontitis, cancers, and inflammatory immune diseases.

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled 16S rRNA-targeting DNA probes has been described. This protocol can be applied to study the role of bacteria in various diseases such as periodontitis, cancers, and inflammatory immune diseases.

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been ...

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

A Method for Targeted 16S Sequencing of Human Milk Samples

Studies of microbial communities have become widespread with the development of relatively inexpensive, rapid, and high throughput sequencing. However, as with all these technologies, reproducible results depend on a laboratory workflow that incorporates appropriate precautions and controls. This is particularly important with low-biomass samples where contaminating bacterial DNA can generate misleading results. This article details a semi-automated workflow to identify microbes from human breast milk samples using targeted sequencing of the 16S ribosomal RNA (rRNA) V4 region on a low- to mid-throughput scale. The protocol describes sample preparation from whole milk including: sample lysis, nucleic acid extraction, amplification of the V4 region of the 16S rRNA gene, and library preparation with quality control measures. Importantly, the protocol and discussion consider issues that are salient to the preparation and analysis of low-biomass samples including appropriate positive and negative controls, PCR inhibitor removal, sample contamination by environmental, reagent, or experimental sources, and experimental best practices designed to ensure reproducibility. While the protocol as described is specific to human milk samples, it is adaptable to numerous low- and high-biomass sample types, including samples collected on swabs, frozen neat, or stabilized in a preservation buffer.

A semi-automated workflow is presented for targeted sequencing of 16S rRNA from human milk and other low-biomass sample types.

Studies of microbial communities have become widespread with the development of relatively inexpensive, rapid, and high throughput sequencing. However, as ...

Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, a significant stress response is induced during gastrointestinal transit and upon bile exposure, a normal component of human digestion. To overcome the bactericidal effects of bile, many enteric pathogens form a biofilm hypothesized to permit survival when transiting through the small intestine. Here we present methodologies to define biofilm formation through solid-phase adherence assays as well as extracellular polymeric substance (EPS) matrix detection and visualization. Furthermore, biofilm dispersion assessment is presented to mimic the analysis of events triggering release of bacteria during the infection process. Crystal violet staining is used to detect adherent bacteria in a high-throughput 96-well plate adherence assay. EPS production assessment is determined by two assays, namely microscopy staining of the EPS matrix and semi-quantitative analysis with a fluorescently-conjugated polysaccharide binding lectin. Finally, biofilm dispersion is measured through colony counts and plating. Positive data from multiple assays support the characterization of biofilms and can be utilized to identify bile salt-induced biofilm formation in other bacterial strains.

This protocol enables the reader to analyze bile salt-induced biofilm formation in enteric pathogens using a multifaceted approach to capture the dynamic nature of bacterial biofilms by assessing adherence, extracellular polymeric substance matrix formation, and dispersion.

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, ...