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A flowchart. A square plate with a grid of circles is labeled grow microorganisms on MALDI-plate. Then irradiate sample. Then use mass spectrometer to measure gaseous ions released. Then compare mass spectrum of sample to reference spectra; two small graphs are shown here. Then identify specied.
MALDI-TOF methods are now routinely used for diagnostic procedures in clinical microbiology laboratories. This technology is able to rapidly identify some microorganisms that cannot be readily identified by more traditional methods. (credit “MALDI plate photo”: modification of work by Chen Q, Liu T, Chen G; credit “graphs”: modification of work by Bailes J, Vidal L, Ivanov DA, Soloviev M)

Microbes can also be identified by measuring their unique lipid profiles. As we have learned, fatty acids of lipids can vary in chain length, presence or absence of double bonds, and number of double bonds, hydroxyl groups, branches, and rings. To identify a microbe by its lipid composition, the fatty acids present in their membranes are analyzed. A common biochemical analysis used for this purpose is a technique used in clinical, public health, and food laboratories. It relies on detecting unique differences in fatty acids and is called fatty acid methyl ester (FAME) analysis . In a FAME analysis , fatty acids are extracted from the membranes of microorganisms, chemically altered to form volatile methyl esters, and analyzed by gas chromatography (GC) . The resulting GC chromatogram is compared with reference chromatograms in a database containing data for thousands of bacterial isolates to identify the unknown microorganism ( [link] ).

A flowchart. A bacterial culture is grown (image is of an agar plate). Then fatty acids are extracted and converted to methyl esters (image is of a test tube). Then gas chromatography analyzes methyl ester fingerprints (image is of a chromatography machine). Then bacteria are identified (image is of a graph). The X axis of the graph is column retention time. The Y axis is of detector signal. The line has various peaks.
Fatty acid methyl ester (FAME) analysis in bacterial identification results in a chromatogram unique to each bacterium. Each peak in the gas chromatogram corresponds to a particular fatty acid methyl ester and its height is proportional to the amount present in the cell. (credit “culture”: modification of work by the Centers for Disease Control and Prevention; credit “graph”: modification of work by Zhang P. and Liu P.)

A related method for microorganism identification is called phospholipid-derived fatty acids (PLFA) analysis . Membranes are mostly composed of phospholipids, which can be saponified (hydrolyzed with alkali) to release the fatty acids. The resulting fatty acid mixture is then subjected to FAME analysis, and the measured lipid profiles can be compared with those of known microorganisms to identify the unknown microorganism.

Bacterial identification can also be based on the proteins produced under specific growth conditions within the human body. These types of identification procedures are called proteomic analysis . To perform proteomic analysis, proteins from the pathogen are first separated by high-pressure liquid chromatography (HPLC) , and the collected fractions are then digested to yield smaller peptide fragments. These peptides are identified by mass spectrometry and compared with those of known microorganisms to identify the unknown microorganism in the original specimen.

Microorganisms can also be identified by the carbohydrates attached to proteins (glycoproteins) in the plasma membrane or cell wall. Antibodies and other carbohydrate-binding proteins can attach to specific carbohydrates on cell surfaces, causing the cells to clump together. Serological tests (e.g., the Lancefield groups tests, which are used for identification of Streptococcus species) are performed to detect the unique carbohydrates located on the surface of the cell.

Resolution

Penny stopped using her new sunscreen and applied the corticosteroid cream to her rash as directed. However, after several days, her rash had not improved and actually seemed to be getting worse. She made a follow-up appointment with her doctor, who observed a bumpy red rash and pus-filled blisters around hair follicles ( [link] ). The rash was especially concentrated in areas that would have been covered by a swimsuit. After some questioning, Penny told the physician that she had recently attended a pool party and spent some time in a hot tub. In light of this new information, the doctor suspected a case of hot tub rash , an infection frequently caused by the bacterium Pseudomonas aeruginosa , an opportunistic pathogen that can thrive in hot tubs and swimming pools, especially when the water is not sufficiently chlorinated. P. aeruginosa is the same bacterium that is associated with infections in the lungs of patients with cystic fibrosis .

The doctor collected a specimen from Penny’s rash to be sent to the clinical microbiology lab. Confirmatory tests were carried out to distinguish P. aeruginosa from enteric pathogens that can also be present in pool and hot-tub water. The test included the production of the blue-green pigment pyocyanin on cetrimide agar and growth at 42 °C. Cetrimide is a selective agent that inhibits the growth of other species of microbial flora and also enhances the production of P. aeruginosa pigments pyocyanin and fluorescein, which are a characteristic blue-green and yellow-green, respectively.

Tests confirmed the presence of P. aeruginosa in Penny’s skin sample, but the doctor decided not to prescribe an antibiotic. Even though P. aeruginosa is a bacterium, Pseudomonas species are generally resistant to many antibiotics. Luckily, skin infections like Penny’s are usually self-limiting; the rash typically lasts about 2 weeks and resolves on its own, with or without medical treatment. The doctor advised Penny to wait it out and keep using the corticosteroid cream. The cream will not kill the P. aeruginosa on Penny’s skin, but it should calm her rash and minimize the itching by suppressing her body’s inflammatory response to the bacteria.

Skin with red raised bumps.
Exposure to Pseudomonas aeruginosa in the water of a pool or hot tub can sometimes cause a skin infection that manifests as “hot tub rash.” (credit: modification of work by “Lsupellmel”/Wikimedia Commons)

Go back to the previous Clinical Focus box.

Key concepts and summary

  • Accurate identification of bacteria is essential in a clinical laboratory for diagnostic and management of epidemics, pandemics, and food poisoning caused by bacterial outbreaks.
  • The phenotypic identification of microorganisms involves using observable traits, including profiles of structural components such as lipids, biosynthetic products such as sugars or amino acids, or storage compounds such as poly-β-hydroxybutyrate.
  • An unknown microbe may be identified from the unique mass spectrum produced when it is analyzed by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF) .
  • Microbes can be identified by determining their lipid compositions, using fatty acid methyl esters ( FAME ) or phospholipid-derived fatty acids ( PLFA ) analysis .
  • Proteomic analysis , the study of all accumulated proteins of an organism; can also be used for bacterial identification.
  • Glycoproteins in the plasma membrane or cell wall structures can bind to lectins or antibodies and can be used for identification.

Fill in the blank

A FAME analysis involves the conversion of _______ to more volatile _____ for analysis using ____________.

fatty acids, methyl esters, gas chromatography

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True/false

MALDI-TOF relies on obtaining a unique mass spectrum for the bacteria tested and then checking the acquired mass spectrum against the spectrum databases registered in the analysis software to identify the microorganism.

True

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Lancefield group tests can identify microbes using antibodies that specifically bind cell-surface proteins.

False

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Short answer

Compare MALDI-TOF, FAME, and PLFA, and explain how each technique would be used to identify pathogens.

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Questions & Answers

characteristic of Gram negative bacteria
jane Reply
Characteristics of Gram Negative Bacteria As with Gram positive bacteria, Gram negative bacteria also contain the peptidoglycan polymer in their cell wall. While this polymer is thin (2 to 4 nanometers in thickness with just about 3 layers of peptidoglycan) in Gram negative bacteria, it's also com
Kaviya
it's also composed of long glycan strands that are cross-linked by peptide molecules. This composition serves a number of functions including protecting the bacterial cell from lysis
Kaviya
Good shot
Enoch
Thanks 😊
Kaviya
what was Hans Christian Gram's supported in the modern Microbiology?
Wilson Reply
what is microbial growth
Chisa Reply
The organism responsible for vulva ulcers
nyiter Reply
Why are vascular pathogen poorly communicable from person to person?
Aj Reply
Most vascular pathogens are poorly communicable from person to person because they need a medium to be communicated i,e a vector that would carry them from one person to other
Kaviya
what's the habit of protista
Afieahngwi Reply
They show both autotrophic and heterotrophic mechanisms...
Swetha
thanks
Afieahngwi
welcome...
Swetha
let me mention some water. Air .Food and so on
Gattiek Reply
causes of infectious diseases
Afieahngwi Reply
water.Air
Gattiek
infectious disease are caused by pathogenic micro organisms like bacteria ,fungi..
Swetha
What is pasteurization?
Wilson
are fungi prokaryote or eukaryotes?
Afieahngwi Reply
fungi are eukaryotes.
Swetha
All fungi are eukaryotes. Even micro fungi.
Lad
have..complex cellular organization and membrane bound nucleus ...and..also... having loops of DNA( like plasmids) as.bacteria
Swetha
what enzyme replaces rna nucleotides with dna nucleotides during replication?
Remi Reply
an enzyme called DNA ligase.
Jael
describe the acid fast staining procedure used in the diagnosis of tuberculosis
Salma Reply
bacterial morphology
lf_ Reply
what is the difference between biogenesis & abiogenesis
Mayuri Reply
biogenesis is when living comes out from other living things as a result of reproduction while a biogenesis is the process where living things comes out from non living things
Usman
living things come form other form living things is biogenesis. ....right?
Mayuri
what is mean by pasturation method?
Mayuri Reply
Pasteurization is a process that kills harmful bacteria and creates an extended shelf life for your milk. ... It's pretty simple—we take the milk from the cows, we rapidly heat it to a high enough temperature to kill the bacteria, and then we cool it back down before packaging and shipping it to you
Kaviya
tell me about abiogenessis &biogenesis
Mayuri
discribe aristol spontaneous generation theory in brif
Mayuri Reply
The Greek philosopher Aristotle (384–322 BC) was one of the earliest recorded scholars to articulate the theory of spontaneous generation, the notion that life can arise from nonliving matter. Aristotle proposed that life arose from nonliving material if the material contained pneuma (“vital heat”).
Kaviya
thank you 😊
Mayuri
No mention dear 😊
Kaviya
Practice MCQ 5

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Source:  OpenStax, Microbiology. OpenStax CNX. Nov 01, 2016 Download for free at http://cnx.org/content/col12087/1.4
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