We thank the technicians of the bacteriology laboratory of the University Hospital Center of Lausanne for their help to implement the techniques in the laboratory.

This protocol has been developed and validated following the research and development processes and ethical rules of our institution before being implemented as a routine tool.
1. Preparation of a Blood Culture Bacterial Pellet by Ammonium Chloride Erythrocyte-lysing Procedure
<ol>
	<li>Preparation of a positive blood culture for subsequent centrifugation.
	<ol>
		<li>Sterilize the blood culture cap. Add 70% ethanol on the cap of the bottle and burn it. 
		NOTE: Do not perform this step under a laminar flow hood.</li>
		<li>Move the bottle to a laminar flow hood. Collect 5 ml of the blood culture with a 20 G syringe.</li>
		<li>Add the 5 ml of blood culture in a 50 ml centrifuge tube containing 45 ml of sterile water (H2O) and mix the sample.</li>
	</ol>
	</li>
	<li>Preparation of a blood culture bacterial pellet by lysis centrifugation.
	<ol>
		<li>Centrifuge the sample at 1,000 x g for 10 min at RT.</li>
		<li>Remove the supernatant (which contains mainly H2O and blood cells) with a laboratory vacuum pump.</li>
		<li>Resuspend the pellet in 1 ml of home-made ammonium chloride lysing solution (0.15 M NH4Cl, 1 mM KHCO3). Break down the pellet by scraping and by vortexing the tube and centrifuge the sample at 140 x g for 10 min at RT.</li>
		<li>Discard the supernatant which mainly contains red blood cells debris.
		<ol>
			<li>If the pellet remained hemorrhagic, resuspend the pellet in 2 ml of sterile and distilled H20 to further lyse residual red blood cells and to wash the pellet. Centrifuge the sample at 140 x g for 10 min at RT and discard the supernatant.</li>
		</ol>
		</li>
		<li>Resuspend the pellet in 200 &#181;l of H2O</li>
	</ol>
	</li>
	<li>Subculture of blood culture on various selective and non-selective agar media.
	<ol>
		<li>Collect 1 ml of the blood culture with a 20 G syringe.</li>
		<li>Inoculate 1 drop of blood culture into four quadrants on blood agar, chocolate agar, McConkey agar and Schaedler agar.</li>
		<li>Incubate at 37 &#176;C the blood agar and chocolate agar plates in a 5% CO2 atmosphere, the McConkey agar in a normal atmosphere and the Schaedler agar plate in anaerobic conditions.</li>
		<li>Follow bacterial growth on the different media.</li>
		<li>Check for bacterial purity.</li>
	</ol>
	</li>
</ol>
2. Identification by MALDI-TOF MS
<ol>
	<li>Direct identification from the blood pellet.
	<ol>
		<li>Transfer 1 &#181;l of the blood pellet on a MALDI target plate and let it dry on a 42 &#176;C heating platform.</li>
		<li>Overlay the sample with 1 &#181;l 70% formic acid and let it dry on a 42 &#176;C heating platform.</li>
		<li>Overlay the sample with 1 &#181;l of MALDI matrix (saturated solution of &#945;-cyano-4-hydroxycinnamic acid in 50% acetonitrile-2.5% trifluoroacetic acid) and let it dry on a 42 &#176;C heating platform.</li>
		<li>Proceed to MALDI-TOF MS analysis with a MALDI-TOF MS system as described by the manufacturers.</li>
		<li>If the identification score is invalid at the species level (for instance with a score &#60; 2), perform a protein extraction of the blood pellet sample.</li>
	</ol>
	</li>
	<li>Identification after protein extraction from the blood culture pellet.
	<ol>
		<li>Mix 20 &#181;l of the pellet with 1 ml of 70% ethanol and centrifuge at 13,000 x g for 2 min at RT.</li>
		<li>Discard the supernatant and add 25 &#181;l of 70% formic acid and 25 &#181;l of 100% acetonitrile to the pellet. Vortex vigorously to resuspend the pellets. Resuspend tough pellets by breaking them down with pipette tips before vortexing.</li>
		<li>Centrifuge at 13,000 x g for 2 min at RT. Transfer 1 &#181;l of the supernatant (which contain extracted proteins) on a MALDI target plate and let it dry on a 42 &#176;C heating platform. Proceed as described in 2.1.2.</li>
	</ol>
	</li>
</ol>
3. Bacterial Identification and Antibiotic Susceptibility Testing with an Automated Microbial System
<ol>
	<li>Preparation of a bacterial suspension for bacterial identification and AST with an automated microbial system.
	<ol>
		<li>Prepare a plastic tube containing 3 ml of sterile 0.45% NaCl solution compatible with the automated microbial system.</li>
		<li>Add a sufficient volume of the blood culture pellet in the 0.45 % NaCl solution to obtain a bacterial suspension corresponding to a McFarland turbidity of 0.6 to 0.8. Measure the turbidity using a densitometer machine. 
		NOTE: The volume of the blood culture bacterial pellet varies from 50 to 200 &#181;l to achieve a McFarland turbidity of 0.6 to 0.8.</li>
	</ol>
	</li>
	<li>Inoculate the automated Gram-positive (GP) or Gram-negative (GN) cards for identification and the automated AST cards for antimicrobial susceptibility testing of Gram-positive cocci and Gram-negative bacteria as described by the manufacturer. 
	NOTE: Identification with GP or GN cards is not applied if MALDI-TOF MS identification score value is &#62;2. Check the purity of the bacterial suspension by subculturing the bacterial suspension on blood agar (GP and GN) and MacConkey agar (GN) plates.</li>
</ol>
4. Antibiotic Susceptibility Testing by Disk Diffusion Assay
The disk diffusion assay is described by the European committee on antimicrobial susceptibility testing (EUCAST, Version 3.0, April 2013, www.eucast.org).
<ol>
	<li>Preparation of a bacterial suspension to perform antimicrobial susceptibility testing by disk diffusion assay.
	<ol>
		<li>Prepare a glass tube containing 3 ml of sterile 0.9% NaCl solution.</li>
		<li>Add a sufficient volume of the blood culture pellet in the 0.9% NaCl solution to obtain a bacterial suspension corresponding to a McFarland turbidity of 0.5. Measure the turbidity using a densitometer machine. 
		NOTE: The volume of the blood culture bacterial pellet varies from 50 to 200 &#181;l to achieve a McFarland turbidity of 0.6 to 0.8.</li>
		<li>Vortex the bacterial suspension.</li>
	</ol>
	</li>
	<li>Inoculation of bacterial suspension onto Mueller-Hinton agar (MH) or Mueller-Hinton agar-fastidious organisms agar (MHF) (MH supplemented with 5% defibrinated horse blood and 20 mg/L of &#946;-Nicotinamide adenine dinucleotide).
	<ol>
		<li>Dip a sterile cotton swab in the bacterial suspension and gently remove excess fluid by turning the swab on the inside wall of the glass tube.</li>
		<li>Spread the bacterial suspension evenly onto the entire surface of the MH/MHF agar plate by swabbing in three directions or by using an automated plate rotator.</li>
	</ol>
	</li>
	<li>Application of antimicrobial disks on inoculated agar plates.
	<ol>
		<li>Apply closely the disks on the surface of the dried inoculated agar plates manually or by using a susceptibility disk dispenser.</li>
		<li>Incubate the plate at the appropriate temperature and atmosphere as described in the antimicrobial susceptibility testing EUCAST disk diffusion method (Version 3.0, April 2013, www.eucast.org).</li>
	</ol>
	</li>
</ol>
5. Automated PCR-based Diagnostic Test for the Detection of MRSA
<ol>
	<li>Using a micropipette, transfer 50 &#181;l of the blood culture pellet into the sample reagent vial provided with the MRSA automated PCR-based diagnostic test kit and vortex during 20 sec.</li>
	<li>Using a 1 ml micropipette, dispense the sample into the specimen port of the cartridge. Insert the cartridge into the automated PCR system and start the assay as described by the manufacturer.</li>
</ol>

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The authors declare that they have no competing financial interests.

Compared to conventional positive blood culture diagnostic approaches, the rapid acquisition of a bacterial pellet by using the ammonium chloride lysis centrifugation approach reduce the time to report identification by 16 to 24 hr and the time to report AST by 24 to 48 hr (Figures 1 and 3).
Rapid introduction of appropriate antibiotic therapy is pivotal to improve the outcome of patients suffering from bloodstream infections. Thus, early identification of the infectious agent...

Bloodstream infections and sepsis in hospitalized patients are a major cause of morbidity and mortality. Thus, mortality related to bloodstream infections is observed in about 14% to 37% of hospitalized patient and may increase to 35% in intensive care units patients 1-3. The rapid identification of the infectious agent is pivotal to guide optimal antimicrobial treatment and to increase the successful outcome of antimicrobial therapy 4,5. The rapid analysis of Gram stains from positive blood culture has already a significant impact on the adaptation of antimicrobial therapy 6,7 but accurate identification of the infectious agent is required to provide the best adapted antibiotic treatment to the patients. For instance, different antibiotic treatment regimens have to be implemented following bacteremia with enterococci and streptococci that are difficult to distinguish by Gram staining. Similarly, identification at the species level is required to detect Gram negative enterobacteria encoding a chromosomal ampC gene which confer an increased resistance to &#946;-lactams 8.
With a positive blood culture, the conventional diagnostic approach is to subculture the infectious agent on different agar plates, which requires several hours of additional incubation prior identification with various approaches including biochemical tests, growth on different selective media and automated microbial identification systems. The time to results of a conventional diagnostic approach is of about 1 to 3 days.
The emergence of the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) technology for rapid identification of microorganisms has provided a new tool to quickly identify microorganisms from colonies grown on agar plates but also directly from positive blood cultures (Figure 1) 9-12. The use of MALDI-TOF to identify an infectious agent from blood cultures has significantly reduced the time to results to a few minutes instead of the hours and days required by traditional methods. As discussed by Croxatto et al. 13, the efficiency of MALDI-TOF identification relies on different parameters including the microorganism&#8217;s purity and quantity. These two criteria are easily obtained from discrete colonies grown on agar plates but required a pre-analytical treatment for bacterial enrichment and purification from complex samples such as blood culture, which contain multiple cellular and protein components that may interfere with MALDI-TOF identification.
Various microorganisms&#8217; isolation methods from blood culture have been used in a number of studies including saponin or other mild detergents method for bacterial extraction 9,14, serum separator method 10, lysis centrifugation methods 12 and commercially solutions such as the sepsityper kit. Our bacteriology diagnostic laboratory has developed a simple blood-culture bacterial pellet preparation based on ammonium chloride erythrocyte-lysis which allow fast identification of bacteria and yeast by MALDI-TOF and automated identification systems (Figure 2) 15. This blood-culture pellet preparation also provide a sample for other direct downstream applications such as Gram staining, automated PCR-based diagnostic tests such as POCT-PCRs for the rapid detection of methicillin-resistant Staphyloccocus aureus (MRSA), and antibiotic susceptibility testing with automated AST systems and/or by disk diffusion assays on agar plates (Figure 3).
In this work, we describe the different steps for the preparation of the blood-culture bacterial pellet as explained by Prod&#8217;hom et al.15 (Figure 4). We will also describe the protocols for three of the main applications that can be performed on the blood culture pellet: Identification by MALDI-TOF 15, identification (ID) and antibiotic susceptibility testing (AST) with the automated systems 16 for Enterobacteriaceae and staphylococci and automated PCR-based diagnostic test for the detection of MRSA 17.

<table><tbody><tr><td>20 G needle</td><td>Terumo, Leuven, Belgium</td><td>NN-2038R</td><td></td></tr><tr><td>50 ml Falcon tube</td><td>BD, Franklin Lakes, NJ, USA</td><td>352070</td><td>50 ml centrifuge tubes</td></tr><tr><td>Ammonium chlorure</td><td>Merck, Darmstadt, Germany</td><td>101145</td><td></td></tr><tr><td>Potassium hydrogen carbonate</td><td>Fluka, St. Louis, MO, USA&amp;nbsp;</td><td>60340</td><td></td></tr><tr><td>Formic acid</td><td>Sigma-Aldrich, St. Louis, MO, USA&amp;nbsp;</td><td>F0507</td><td>Flammable, corrosive</td></tr><tr><td>&amp;#945;-Cyano-4-hydroxycinnamic acid</td><td>Fluka, St. Louis, MO, USA&amp;nbsp;</td><td>70990</td><td>Acute toxicity</td></tr><tr><td>Acetonitrile</td><td>Sigma-Aldrich, St. Louis, MO, USA&amp;nbsp;</td><td>271004</td><td>Flammable, acute toxicity</td></tr><tr><td>Trifluoroacetic acid</td><td>Sigma-Aldrich, St. Louis, MO, USA&amp;nbsp;</td><td>T6508</td><td>Corrosive, acute toxicity</td></tr><tr><td>Vitek 2 60 instrument</td><td>Biom&amp;eacute;rieux, Marcy-l&#039;Etoile, France</td><td>27202</td><td>automated microbial system instrument</td></tr><tr><td>Vitek 2 Gram-positive (GP) card</td><td>Biom&amp;eacute;rieux, Marcy-l&#039;Etoile, France</td><td>21342</td><td>automated GP&amp;nbsp; identification card</td></tr><tr><td>Vitek 2 AST-P580 card</td><td>Biom&amp;eacute;rieux, Marcy-l&#039;Etoile, France</td><td>22233</td><td>automated microbial AST system</td></tr><tr><td>Vitek 2 Gram-negative (GN) card</td><td>Biom&amp;eacute;rieux, Marcy-l&#039;Etoile, France</td><td>21341</td><td>automated GN&amp;nbsp; identification card</td></tr><tr><td>Vitek 2 AST-N242 card</td><td>Biom&amp;eacute;rieux, Marcy-l&#039;Etoile, France</td><td>413391</td><td>automated microbial AST system</td></tr><tr><td>Xpert MRSA</td><td>Cepheid, Sunnyvale, Ca, USA</td><td>&amp;nbsp;GXMRSA-100N-10</td><td>nucleic acid amplification technology MRSA</td></tr><tr><td>GeneXpert IV instrument</td><td>Cepheid, Sunnyvale, Ca, USA</td><td>GXIV-4-D</td><td>nucleic acid amplification technology instrument</td></tr><tr><td>Microflex LT MALDI-TOF MS instrument</td><td>Bruker Daltonics, Bremen, Germany</td><td>BDAL microflex LT/SH</td><td></td></tr><tr><td>MSP 96 target steel BC</td><td>Bruker Daltonics, Bremen, Germany</td><td>280799</td><td>MALDI target plate</td></tr><tr><td>Densitometer Densicheck instrument</td><td>Biom&amp;eacute;rieux, Marcy-l&#039;Etoile, France</td><td>27208</td><td></td></tr><tr><td>MALDI Sepsityper kit 50</td><td>Bruker Daltonics, Bremen, Germany</td><td>8270170</td><td></td></tr><tr><td>Mac Conkey agar</td><td>Biolife, Milano, Italy</td><td>4016702</td><td></td></tr><tr><td>Mueller-Hinton agar</td><td>Oxoid, Hampshire, England</td><td>CM0337</td><td>Mueller-Hinton agar (MH)&amp;nbsp;</td></tr><tr><td>MHF agar</td><td>Biom&amp;eacute;rieux, Marcy-l&#039;Etoile, France</td><td>43901</td><td>Mueller-Hinton agar-fastidious organisms agar (MHF)</td></tr><tr><td>BD columbia III agar</td><td>BD, Franklin Lakes, NJ, USA</td><td>254071</td><td>blood agar</td></tr><tr><td>BD chocolate agar</td><td>BD, Franklin Lakes, NJ, USA</td><td>254089</td><td>chocolate agar</td></tr><tr><td>BD schaedler agar</td><td>BD, Franklin Lakes, NJ, USA</td><td>254084</td><td>Schaedler agar</td></tr></tbody></table>

preparation of a blood culture pellet for rapid bacterial identification and antibiotic susceptibility testing

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.

In the study performed by Prod’hom et al.15, bacterial pellets obtained by ammonium chloride lysis centrifugation of 122 positive blood culture from 78 patients were analyzed by MALDI-TOF MS. Out of 122 positive blood culture, 95 (77.9%) were correctly identified at the species level and one (0.8%) at the genus level. The remaining 26 (21.3%) blood culture pellets gave no reliable identification by MALDI-TOF. Among those, 21 were gram positive bacteria including 13 streptococci and 5 coagulase-negat...

Watch this Scientific Journal Video about Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing at JoVE.com

Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

A rapid bacterial pellet preparation from a positive blood culture can be used as a sample for applications such as identification by MALDI-TOF, Gram staining, antibiotic susceptibility testing and PCR-based test. The results can be rapidly communicated to clinicians to improve the outcome of patients suffering from bloodstream infections.

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a ...

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JoVE Journal

Immunology and Infection

35.5K Views.  University Hospital Center and University of Lausanne.  Een snelle bacteriële pellet bereiding van een positieve bloedkweek kan worden gebruikt als een monster voor toepassingen zoals identificatie door MALDI-TOF, Gram-kleuring, antibiotica gevoeligheidstesten en PCR-gebaseerde test. De resultaten kunnen snel worden meegedeeld aan artsen om de uitkomst van patiënten die lijden aan infecties in het bloed te verbeteren. 

Video: Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium. Because resistance to ampicillin and vancomycin in E. faecalis is still uncommon compared to resistance in E. faecium, the development of rapid tests allowing differentiation between enterococcal species is important for appropriate therapy and resistance surveillance. The E. faecalis OE PNA FISH assay (AdvanDx, Woburn, MA) uses species-specific peptide nucleic acid (PNA) probes in a fluorescence in situ hybridization format and offers a time to results of 1.5 hours and the potential of providing important information for species-specific treatment. Multicenter studies were performed to assess the performance of the 1.5 hour E. faecalis/OE PNA FISH procedure compared to the original 2.5 hour assay procedure and to standard bacteriology methods for the identification of enterococci directly from a positive blood culture bottle.

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

A rapid protocol for the direct identification of Enterococcus faecalis and other Enterococcus species from a positive blood culture using a Peptide Nucleic Acid fluorescent in situ hybridization assay (PNA FISH).

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium.  Because resistance to ampicillin and ...

One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. Therefore, rapid administration of adequate antibiotic therapy is of foremost importance in the treatment of bloodstream infections. The critical element in this process is timing, heavily dependent on the results of bacterial identification and antibiotic susceptibility testing. Both of these parameters are routinely obtained by culture-based testing, which is time-consuming and takes on average 24-48 hours2, 4. The aim of the study was to develop DNA-based assays for rapid identification of bloodstream infections, as well as rapid antimicrobial susceptibility testing. The first assay is a eubacterial 16S rDNA-based real-time PCR assay complemented with species- or genus-specific probes5. Using these probes, Gram-negative bacteria including Pseudomonas spp., Pseudomonas aeruginosa and Escherichia coli as well as Gram-positive bacteria including Staphylococcus spp., Staphylococcus aureus, Enterococcus spp., Streptococcus spp., and Streptococcus pneumoniae could be distinguished. Using this multiprobe assay, a first identification of the causative micro-organism was given after 2 h.
Secondly, we developed a semi-molecular assay for antibiotic susceptibility testing of S. aureus, Enterococcus spp. and (facultative) aerobe Gram-negative rods6. This assay was based on a study in which PCR was used to measure the growth of bacteria7. Bacteria harvested directly from blood cultures are incubated for 6 h with a selection of antibiotics, and following a Sybr Green-based real-time PCR assay determines inhibition of growth. The combination of these two methods could direct the choice of a suitable antibiotic therapy on the same day (Figure 1). In conclusion, molecular analysis of both identification and antibiotic susceptibility offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections.

The design of a straightforward one-day workflow scheme for bacterial pathogen diagnostics enables the rapid recognition of bloodstream infections. The inclusion of eight clinically relevant bacterial targets and their antibiotic resistance profiles offers the clinician an initial insight on the same day, which can lead to more adequate therapy.

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. ...

Detection of Bacteria Using Fluorogenic DNAzymes

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses each and every year. Prevention of such outbreaks and minimization of the impact of an ongoing epidemic place an ever-increasing demand for analytical methods that can accurately identify culprit pathogens at the earliest stage. Although there is a large array of effective methods for pathogen detection, none of them can satisfy all the following five premier requirements embodied for an ideal detection method: high specificity (detecting only the bacterium of interest), high sensitivity (capable of detecting as low as a single live bacterial cell), short time-to-results (minutes to hours), great operational simplicity (no need for lengthy sampling procedures and the use of specialized equipment), and cost effectiveness. For example, classical microbiological methods are highly specific but require a long time (days to weeks) to acquire a definitive result.1 PCR- and antibody-based techniques offer shorter waiting times (hours to days), but they require the use of expensive reagents and/or sophisticated equipment.2-4 Consequently, there is still a great demand for scientific research towards developing innovative bacterial detection methods that offer improved characteristics in one or more of the aforementioned requirements. Our laboratory is interested in examining the potential of DNAzymes as a novel class of molecular probes for biosensing applications including bacterial detection.5
DNAzymes (also known as deoxyribozymes or DNA enzymes) are man-made single-stranded DNA molecules with the capability of catalyzing chemical reactions.6-8 These molecules can be isolated from a vast random-sequence DNA pool (which contains as many as 1016 individual sequences) by a process known as "in vitro selection" or "SELEX" (systematic evolution of ligands by exponential enrichment).9-16 These special DNA molecules have been widely examined in recent years as molecular tools for biosensing applications.6-8
Our laboratory has established in vitro selection procedures for isolating RNA-cleaving fluorescent DNAzymes (RFDs; Fig. 1) and investigated the use of RFDs as analytical tools.17-29 RFDs catalyze the cleavage of a DNA-RNA chimeric substrate at a single ribonucleotide junction (R) that is flanked by a fluorophore (F) and a quencher (Q). The close proximity of F and Q renders the uncleaved substrate minimal fluorescence. However, the cleavage event leads to the separation of F and Q, which is accompanied by significant increase of fluorescence intensity.
More recently, we developed a method of isolating RFDs for bacterial detection.5 These special RFDs were isolated to "light up" in the presence of the crude extracellular mixture (CEM) left behind by a specific type of bacteria in their environment or in the media they are cultured (Fig. 1). The use of crude mixture circumvents the tedious process of purifying and identifying a suitable target from the microbe of interest for biosensor development (which could take months or years to complete). The use of extracellular targets means the assaying procedure is simple because there is no need for steps to obtain intracellular targets.
Using the above approach, we derived an RFD that cleaves its substrate (FS1; Fig. 2A) only in the presence of the CEM produced by E. coli (CEM-EC).5 This E. coli-sensing RFD, named RFD-EC1 (Fig. 2A), was found to be strictly responsive to CEM-EC but nonresponsive to CEMs from a host of other bacteria (Fig. 3).
Here we present the key experimental procedures for setting up E. coli detection assays using RFD-EC1 and representative results.

We have recently reported a novel approach for generating fluorogenic DNAzyme probes that can be applied to set up a simple, "mix-and-read" fluorescent assay for bacterial detection. These special DNA probes catalyze the cleavage of a chromophore-modified DNA-RNA chimeric substrate in the presence of crude extracellular mixture (CEM) produced by a specific bacterium, thereby translating bacterial detection into fluorescence signal generation. In this report we will describe key experimental procedures where a specific DNAzyme probe denoted "RFD-EC1" is employed for the detection of the model bacterium, Escherichia coli (E. coli).

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses ...

Isolation and Identification of Waterborne Antibiotic-Resistant Bacteria and Molecular Characterization of their Antibiotic Resistance Genes

The development and spread of antibiotic resistance (AR) through microbiota associated with freshwater bodies is a major global health concern. In the present study, freshwater samples were collected and analyzed with respect to the total bacterial diversity and AR genes (ARGs) using both conventional culture-based techniques and a high-throughput culture-independent metagenomic approach. This paper presents a systematic protocol for the enumeration of the total and antibiotic-resistant culturable bacteria from freshwater samples and the determination of phenotypic and genotypic resistance in the culturable isolates. Further, we report the use of whole metagenomic analysis of the total metagenomic DNA extracted from the freshwater sample for the identification of the overall bacterial diversity, including non-culturable bacteria, and the identification of the total pool of different ARGs (resistome) in the water body. Following these detailed protocols, we observed a high antibiotic-resistant bacteria load in the range of 9.6 &#215; 105-1.2 &#215; 109 CFU/mL. Most isolates were resistant to the multiple tested antibiotics, including cefotaxime, ampicillin, levofloxacin, chloramphenicol, ceftriaxone, gentamicin, neomycin, trimethoprim, and ciprofloxacin, with multiple antibiotic resistance (MAR) indexes of &#8805;0.2, indicating high levels of resistance in the isolates. The 16S rRNA sequencing identified potential human pathogens, such as Klebsiella pneumoniae,&#160;and opportunistic bacteria, such as Comamonas spp., Micrococcus spp., Arthrobacter spp., and Aeromonas spp. The molecular characterization of the isolates showed the presence of various ARGs, such as blaTEM, blaCTX-M (&#946;-lactams), aadA, aac (6&#39;)-Ib (aminoglycosides), and dfr1 (trimethoprims), which was also confirmed by the whole metagenomic DNA analysis. A high prevalence of other ARGs encoding for antibiotic efflux pumps-mtrA, macB, mdtA, acrD, &#946;-lactamases-SMB-1, VIM-20, ccrA, ampC, blaZ, the&#160;chloramphenicol acetyltransferase gene catB10, and&#160;the rifampicin resistance gene rphB-was also detected in the metagenomic DNA. With the help of the protocols discussed in this study, we confirmed the presence of waterborne MAR bacteria with diverse AR phenotypic and genotypic traits. Thus, whole metagenomic DNA analysis can be used as a complementary technique to conventional culture-based techniques to determine the overall AR status of a water body.

Here, we present a detailed protocol for the isolation and identification of antibiotic-resistant bacteria from water and the molecular characterization of their antibiotic resistance genes (ARGs). The use of culture-based and non-culture-based (metagenomic analysis) techniques provides complete information about the total bacterial diversity and the total pool of different ARGs present in freshwaters from Mumbai, India.

The development and spread of antibiotic resistance (AR) through microbiota associated with freshwater bodies is a major global health concern. In the ...

Environment

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 ...

Antimicrobial Synergy Testing by the Inkjet Printer-assisted Automated Checkerboard Array and the Manual Time-kill Method

As rates of multidrug-resistant (MDR) pathogens continue to rise, outpacing the development of new antimicrobials, novel approaches to treatment of MDR bacteria are increasingly becoming a necessity. One such approach is combination therapy, in which two or more antibiotics are used together to treat an infection against which one or both of the drugs may be ineffective alone. When two drugs, in combination, exert a greater than additive effect, they are considered synergistic. In vitro investigation of synergistic activity is an important first step in evaluating the possible efficacy of drug combinations. Two main in vitro synergy testing methods have been developed: the checkerboard array and the time-kill study. In this paper, we present an automated checkerboard array method that makes use of inkjet printing technology to increase the efficiency and accuracy of this technique, as well as a standard manual time-kill synergy method. The automated checkerboard array can serve as a high-throughput screening assay, while the manual time-kill study provides additional, complementary data on synergistic activity and killing.
The checkerboard array is a modification of standard minimum inhibitory concentration (MIC) testing, in which bacteria are incubated with antibiotics at different concentration combinations and evaluated for growth inhibition after overnight incubation. Manual performance of the checkerboard array requires a laborious and error-prone series of calculations and dilutions. In the automated method presented here, the calculation and dispensing of required antibiotic stock solution volumes are automated through the use of inkjet printer technology. In the time-kill synergy assay, bacteria are incubated with the antibiotics of interest, both together and individually, and sampled at intervals over the course of 24 h for quantitative culture. The results can determine whether a combination is synergistic and whether it is bactericidal, and provide data on inhibition and killing of bacteria over time.

Antimicrobial synergy testing is used to evaluate the effect of two or more antibiotics used in combination and is typically performed by one of two methods: the checkerboard array or the time-kill assay. Here, we present an automated, inkjet printer-assisted checkerboard array synergy technique and a classic time-kill synergy study.

As rates of multidrug-resistant (MDR) pathogens continue to rise, outpacing the development of new antimicrobials, novel approaches to treatment of MDR ...

Sorting of Streptomyces Cell Pellets Using a Complex Object Parametric Analyzer and Sorter

Streptomycetes are filamentous soil bacteria that are used in industry for the production of enzymes and antibiotics. When grown in bioreactors, these organisms form networks of interconnected hyphae, known as pellets, which are heterogeneous in size. Here we describe a method to analyze and sort mycelial pellets using a Complex Object Parametric Analyzer and Sorter (COPAS). Detailed instructions are given for the use of the instrument and the basic statistical analysis of the data. We furthermore describe how pellets can be sorted according to user-defined settings, which enables downstream processing such as the analysis of the RNA or protein content. Using this methodology the mechanism underlying heterogeneous growth can be tackled. This will be instrumental for improving streptomycetes as a cell factory, considering the fact that productivity correlates with pellet size.

Sorting of Streptomyces Cell Pellets Using a Complex Object Parametric Analyzer and Sorter

Liquid-grown Streptomyces cultures are characterized by mycelial pellets that are heterogeneous in size. We here describe a method to analyze and sort such pellets in a high-throughput manner. These pellets can be used for further analyses, which will provide leads to understand and control growth heterogeneity.

Streptomycetes are filamentous soil bacteria that are used in industry for the production of enzymes and antibiotics. When grown in bioreactors, these ...

Biology

Accelerated Antimicrobial Susceptibility Testing in Blood Culture Bottles

Direct Microbial Identification using An Automated Microbial Identification System to Facilitate the EUCAST RAST Method Without Mass Spectrometry

Gram-negative (GN) sepsis is a medical emergency where management in resource-limited settings relies on conventional microbiological culture techniques providing results in 3-4 days. Recognizing this delay in turnaround time (TAT), both EUCAST and CLSI have developed protocols for determining AST results directly from positively flagged automated blood culture bottles (+aBCs). EUCAST rapid AST (RAST) protocol was first introduced in 2018, where zone diameter breakpoints for four common etiological agents of GN sepsis, i.e., Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii complex can be reported. However, those clinical laboratories that have implemented this method in their routine workflow rely on mass spectrometry-based microbial identification, which is not easily available, thus precluding its implementation in resource-limited settings. To circumvent it, we evaluated a direct inoculum protocol (DIP) using a commercial automated microbial identification and antimicrobial susceptibility testing system (aMIAST) to enable early microbial identification within 8 h of positive flagging of aBC. We evaluated this protocol from January to October 2023 to identify the four RAST reportable GN (RR-GN) in the positively flagged aBC. The microbial identification results in DIP were compared with the standard inoculum preparation protocol (SIP) in aMIAST. Of 204 +aBCs with monomorphic GN (+naBC), one of the 4 RR-GN was identified in 105 +naBCs by SIP (E. coli: 50, K. pneumoniae: 20, P. aeruginosa: 9 and A. baumannii complex: 26). Of these, 94% (98/105) were correctly identified by DIP whereas major error and very major error rates were 6% (7/105) and 1.7% (4/240), respectively. When DIP for microbial identification is done using the EUCAST RAST method, provisional clinical reports can be provided within 24 h of receiving the sample. This approach has the potential to significantly reduce the TAT, enabling early institution of appropriate antimicrobial therapy.

Author Spotlight: Accelerating Diagnostic Accuracy with Direct Identification of Gram-Negatives from Blood Culture Bottles

EUCAST has developed a direct antimicrobial susceptibility testing (AST) protocol for automated blood cultures. However, its dependence on mass spectrometry-based microbial identification can be obviated by using a direct inoculum preparation protocol in an automated microbial identification system. This approach can provide AST reports within 24 h of sample collection.

Gram-negative (GN) sepsis is a medical emergency where management in resource-limited settings relies on conventional microbiological culture techniques ...

Medicine

Rapid Antimicrobial Susceptibility Testing by Stimulated Raman Scattering Imaging of Deuterium Incorporation in a Single Bacterium

To slow and prevent the spread of antimicrobial resistant infections, rapid antimicrobial susceptibility testing (AST) is in urgent need to &#65279;quantitatively determine the &#65279;antimicrobial effects on pathogens. &#65279;It typically takes days to complete the AST by conventional methods based on the long-time culture, and they do not work directly for clinical samples. Here, we report a rapid AST &#65279;method enabled by stimulated Raman scattering (SRS) imaging of deuterium oxide (D2O) metabolic incorporation. Metabolic incorporation of D2O into biomass and the metabolic activity inhibition upon exposure to antibiotics at the single bacterium level are monitored by SRS imaging. The&#65279; single-cell metabolism inactivation concentration (SC-MIC) of bacteria upon exposure to antibiotics can be obtained after a total of 2.5 h of sample preparation and detection. Furthermore, this rapid AST method is directly applicable to bacterial samples in complex biological environments, such as urine or whole blood. SRS metabolic imaging of deuterium incorporation is transformative for rapid single-cell phenotypic AST in the clinic.

This protocol presents rapid antimicrobial susceptibility testing (AST) assay within 2.5 h by single-cell-stimulated Raman scattering imaging of D2O metabolism. This method applies to bacteria in the urine or whole blood environment, which is transformative for rapid single-cell phenotypic AST in the clinic.

To slow and prevent the spread of antimicrobial resistant infections, rapid antimicrobial susceptibility testing (AST) is in urgent need to ...

Pure Cultures and Streak Plating: Isolation of Single Bacterial Colonies from a Mixed Sample

Source: Tilde Andersson1, Rolf Lood1 
1 Department of Clinical Sciences Lund, Division of Infection Medicine, Biomedical Center, Lund University, 221 00 Lund, Sweden
Seemingly impossible to determine, microbial biodiversity is truly astounding with an estimated one trillion coexisting species (1,2). Although particularly harsh climates, like the acidic environment of the human stomach (3) or the subglacial lakes of Antarctica (4), may be dominated by a specific species, bacteria are typically found in mixed cultures. As each strain may influence the growth of another (5), the ability to separate and cultivate &#34;pure&#34; (consisting only of one type) colonies has become essential in clinical and academic settings alike. Pure cultures enable further genetic (6) and proteomic examinations (7), analysis of sample purity and, perhaps more noteworthy, the identification and characterization of infectious agents from clinical samples.
Bacteria have a wide range of growth requirements and there are numerous types of nutrient media designed to sustain both the un-demanding and the fastidious species (8). Growth media can be prepared either in liquid form (as a broth) or in a typically agar-based (a gelling agent derived from red algae) solid form. Whereas direct inoculation into broth carries the risk of generating a genetically diverse or even mixed bacterial population, plating and re-streaking creates a purer culture where each cell has a highly similar genetic makeup. The streak plate technique is based on progressive dilution of a sample (Figure 1), with the aim of separating individual cells from one another. Any viable cell (hereafter referred to as a colony forming unit, CFU) sustained by the media and designated environment can subsequently found an isolated colony of daughter-cells through binary fission. In spite of the rapid mutation rates within bacterial communities, this cell-group is generally regarded as clonal. Harvesting and re-streaking this population consequently ensures that subsequent work involves only a single bacterial type.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10509/10509fig1.jpg" /> 
Figure 1: A streak plate is based on progressive dilution of the original sample. I) The inoculum is initially dispersed using a zig-zag motion, creating an area with a relatively dense bacterial population. II-IV) Streaks are drawn from the preceding area, using a sterile inoculation loop each time, until the fourth quadrant is reached. V) A final zig-zag motion directed towards the middle of the plate forms a region where the inoculum has been markedly diluted, allowing colonies to appear separate from one another.
The streak plate technique can also be combined with the use of selective and/or differential media. A selective medium will inhibit the growth of certain organisms (e.g. through addition of antibiotics) whilst a differential medium will solely help distinguish one from another (e.g. through hemolysis on blood agar plates).
Underlying all work in microbiology is the use of aseptic (sterile) techniques. Every bacterial culture should be considered potentially pathogenic as there is a risk of unintended growth of treacherous strains, aerosol formation and contamination of equipment/personnel. To minimize these risks, all media, plastic-, metal- and glass-ware are typically sterilized through autoclaving before and after use, subjecting them to high-pressure saturated steam at around 121&#176;C that effectively wipes out any lingering cells. The work space is generally disinfected using ethanol both prior to, and after, use. Lab coat and gloves are always worn during work with infectious agents.

Pure Cultures and Streak Plating: Isolation of Bacterial Colonies

Source: Tilde Andersson1, Rolf Lood1
1 Department of Clinical Sciences Lund, Division of Infection Medicine, Biomedical Center, Lund University, 221 00 ...

Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

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