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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

deffination of staining
Bhavanimangali Reply
with the aid of a well labeled diagram describe the conducting system
Maridad Reply
what is cellular immunity
namugenyi Reply
Cellular Immunity. -Lymphocytes act against target cell. -Acts directly by killing infected cells.
abdinor
What are NK cells
Peter
Natural killer cells
Rahaba
what are Antigen determinant
mary
cellular immunity is the state where the lymphocytes destroy the infected or targeted cell
cynthia
any examples of oedema
cynthia
introduction of microbial diversity-1
Bhavanimangali Reply
List the type of micro organism arround us and how they can be seen and with what kind of instrument
clinton Reply
how is the arrangements of bacteria in bacilli
Vaidah Reply
Provide some examples of bacterial structures that might be used as antibiotic targets and explain why.
Vaidah
Coccobacilli, Club-Shaped bacilli, Bacilli with rounded ends, Fuilform bacilli, Bacilli with ends square.
Enoch
three main antibiotic targets in bacteria: The cell wall or membranes that surrounds the bacterial cell. The machineries that make the nucleic acids DNA and RNA. The machinery that produce proteins
Rushikesh
The bacterial cell wall. Protein production. and DNA synthesis. Why, this is because most drugs (antibiotics) affects the cell wall of the bacteria, which makes the bacteria weak or susceptible in human body.
Enoch
UV rays affecting the..
Mali Reply
what is microbiology
Baba Reply
Microbiology is the study of microorganisms, which are prokaryotic and eukaryotic cell which includes bacteria, fungi, viruses and pathogenic protozoa.
Enoch
Microbiology is the branch of Life science which deals with scientific study of many Microorganisms.
Rushikesh
what is types of microbiology
Alsheikh
Immunology, Bacteriology, Virology, Mycology, Algology etc
Enoch
Virology, Immunology, Bacteriology, Algology, Mycology, Protoozology etc
Enoch
and what is mycology
Alsheikh
Immunology, Serology, Virology, Microbial Genetics, Parasitology, Bacteriology, Mycology, Molecular, Cell Biology, Agricultural, Water,Soil, Food Industrial ,Pharmaceutical, Applied, Environmental, Clinical, Medical,Marine Microbiology, Microbial Systematics, Etc, are & many types of Microbiology.
Rushikesh
study of fungi is called mycology
Munna
Mycology is the branch of Microbiology which deals with scientific study of Fungi.
Rushikesh
Study of microorganisms,which we can't see with our naked eye is called microbiology
Munna
Mycology is the scientific study of Fungi.
Enoch
virology is the study of viruses
Oppah
what is microbiology? microbiology is the study of small microorganisms that we can not with our naked eyes.
Leticia
what is taxonomical classification of microbiology
Bami
The algae, protozoa, slime moulds, fungi, bacteria, archaea and viruses ,are taxonomic classification of Microorganisms
Rushikesh
We have Bacteria, Archaea, Protozoa, Algae, Fungi, Viruses.
Enoch
microbiology is the study of microbes too small to be seen by naked eyes
Maridad
microbiology is a branch of biology which deals with study of smallest living microrganisms such as bacteria protozoa fungi and viruses
Chaitra
microbiology is the study of microorganisms which can't be seen by our naked eyes
Nakaweesi
Micro - Minute Bio - Life Logus - Study
Roshan
what is the meaning of antimicrobial susceptibility testing
Devshree Reply
seven gram positive bacteria
Okocha Reply
seven examples of gram negative bacteria
Okocha
seven examples of gram negative bacteria
Okocha
Physical conditions that would enable selective Isolation of staphylococcus epidermis
shongile Reply
Nutritional requirements that would enable selective Isolation of staphylococcus epidermis
shongile
Nutritional requirements that would enable selective Isolation of staphylococcus epidermis
shongile
what is constant flux but
Jane Reply
Digestion of food is completed in __
Amina Reply
Small Intestine
Enoch
large inteatine
Abdiwali
small intestine
Betelhem
Small intestine
Abdirisaq
small intestine
Marah
small intestine specific in illum
Mariam
difference btwn hausteria and appears
Raviha Reply
numerical and molecular taxanomy
Dhanshri Reply
difference btwn hausteria and appesorium
Raviha
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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