Columbia agar with colistin and nalidixic acid (CNA) is a selective and differential medium for isolating and differentiating pathogenic gram-positive cocci from clinical and non-clinical samples. Antimicrobials colistin (C) and nalidixic acid (NA) inhibit gram-negative organisms.

Colistin, a polypeptide antibiotic of the polymyxin group, disrupts the cell membranes of gram-negative organisms. Nalidixic acid, a first-generation quinolone, blocks DNA replication in susceptible gram-negative bacteria.

Columbia agar base is a nutritionally rich formula containing three peptone sources and 5% defibrinated (whole blood with fibrin removed to prevent clotting) sheep blood. This supportive medium is used to differentiate bacterial colonies based on the hemolytic reactions.

Gram-negative bacteria such as members of Enterobacteriaceae and Pseudomonas species are suppressed, while staphylococci, streptococci, enterococci, and yeast grows on Columbia CNA agar.

Composition

Columbia CNA agar is prepared by adding 10 mg/liter of colistin and 15 mg/liter of nalidixic acid in Columbia Agar Base.

Columbia agar base is a nutritionally rich medium containing 5% defibrinated blood. Sheep blood provides X and V factors for the growth of fastidious gram-positive bacteria and helps differentiate them based on hemolytic reactions.

Peptone supports the luxuriant growth of microorganisms. Cornstarch serves as a neutralizer source. The composition of Columbia CNA agar is as follows*;

Product	Gram/Liter
Casein-Meat Peptone	10 g
Casein-Yeast Peptone	10 g
Heart Peptone	3 g
Sodium chloride	5 g
Corn starch	1 g
Agar	13.5 g
Defibrinated sheep blood	50 mL
Colistin sulfate	10 mg
Nalidixic Acid	15 mg
pH: 7.3 +/- 0.2

*Variations in the composition may be seen in media prepared by different manufacturers.

Preparation of Media

1. Suspend 44.02 grams in 1000 ml purified/distilled water.
2. Heat to boiling to dissolve the medium completely. Do not overheat the medium.
3. Sterilize by autoclaving at 15 lbs pressure at 121 for 15 minutes.
4. Cool to 45-50°C and aseptically add 5% v/v sterile, defibrinated blood.
5. Mix well and pour into sterile Petri plates (20-25 ml per plate in 90 mm Petri plate).

Storage and Shelf Life

Columbia CNA Agar should be protected from light and stored at 4°C to 8°C.

The medium side should be uppermost to prevent excessive moisture accumulation on the agar surface. Under these conditions, the medium has an 8-week shelf life from the date of manufacture.

Quality Control

After checking for correct pH, color, depth, and sterility, the following organisms are used to determine the growth performance of the completed medium.

Organism	Expected Result
Streptococcus pyogenes	Growth, β-hemolysis
Streptococcus pneumoniae	Growth, α-hemolysis
Staphylococcus aureus	Growth β-hemolysis or no-hemolysis
Proteus mirabilis	Partial inhibition

Inoculation of the Medium

1. Allow the medium to reach room temperature before inoculation.
2. Using a direct inoculum from the specimen, perform a four-quadrant streak to obtain well-isolated colonies. If desired, stabs into the medium can also be made to improve and better observe hemolytic reactions.
3. Depending on the organism of interest, incubate plates aerobically, anaerobically, or in a CO2-enriched environment at 35°C. The hemolytic reaction of some streptococci is influenced by the atmospheric environment of the incubator. For optimal results, culture plates are incubated in a 5% CO2-enriched environment.
4. Examine after 24 and 48 hours.

Interpretation of Results

After incubation, examine plates for growth and hemolysis. To observe hemolysis, view the culture plates with a bright light transmitting behind the plate. Four types of hemolysis can be seen in Columbia CNA Agar:

1. Alpha-hemolysis: Partial hemolysis results in a greenish discoloration around the colony.
2. Beta-hemolysis: Complete lysis of red blood cells resulting in a clear zone around the colonies.
3. Gamma-hemolysis: No hemolysis resulting in no change in the medium.
4. Alpha-prime-hemolysis: A small zone of complete hydrolysis surrounded by an area of partial hemolysis.

Uses

On conventional blood agar, gram-negative bacilli may outgrow gram-positive cocci. Columbia CNA agar helps to isolate gram-positive cocci (Streptococcus species and Staphylococcus species) when present in a mix-culture with gram-negative bacilli. The addition of sheep blood helps to differentiate species of Streptococcus as well.

References

• Bailey & Scott’s Diagnostic Microbiology, Forbes, 11th edition
• Koneman’s Color Atlas and Textbook of Diagnostic Microbiology (Color Atlas & Textbook of Diagnostic Microbiology), 7th edition

Agar (agar agar) or bacteriological agar is a thermoreversible gelling agent extracted from the cell walls of smaller seaweeds (red algae). Agar is obtained from red algae belonging to the genera Gracilaria, Ahnfeltia, Gelidium, and Pterocladiella. 

Bacteriological agar has no taste or smell and is often added to food with other ingredients. About 90% of agar is used for food applications such as bakery bread, ice cream, meringue, fruit puddings, jams, and marmalade. In microbiology laboratories, agar is commonly used to solidify culture media.

Bacteriological Agar 

Bacteriological agar is a hydrophilic colloidal substance made from cell wall components of Gelidium and Rhodoyceae (marine algae) species. Agar is used to solidifying culture media because of its high gelling strength; a setting temperature of 32-39°C, and a melting temperature of 90-95°C. Low gelling temperature (34-36°C) allows the addition of heat-sensitive nutrients such as whole blood to be added safely at 45-50°C with a minimum risk of heat damage.  

Most agars used in bacteriological work produce a firm gel at an agar concentration of 1.5% w/v. At a concentration of 0.4-0.5% w/v, agar gives semisolid gel, which is used to make biochemical media and transport media such as  Amies medium.

As agar media are used to grow bacteria, “Bacto” agars must not contain trace metals and other materials that might inhibit the growth of bacteria or fungi. The gels must be strong and have good clarity. 

Bacteriological agar is obtained from sea algae at only a few harvesting sites and requires rigorous processing to remove naturally occurring pigments, salts, miscellaneous inhibitory substances, and bacterial spores. Manufacturers of bacteriological agar keep all processing details confidential. The increasing use of agar is pushing its cost, and also, there are shortages. So companies are searching for cheaper alternatives to agar to solidify culture media.

Usage History 

Angelina Fanny Hesse (1850-1934) was the first to propose agar use in culture media. She is the wife of one of Robert Koch’s colleagues, Walther Hesse. Ironically, neither Lina nor Walter Hesse was given credit for using agar in microbiology.

Fanny Hesse suggested Robert  Koch to add agar to his bacteriological media. The use of agar created a firm surface over which microorganisms could be spread very thinly, so thinly that some individual organisms were separated from all others. Robert Koch used agar to isolate Mycobacterium tuberculosis.

Chemical Nature

Agar (also called agar-agar) is a mixture of polysaccharides whose basic monomer is galactose. Agar consists of two fractions, agarose and agaropectin. Agarose is a linear polysaccharide and the gel-forming component; agaropectin is a branched, nongelling component of agar. 

Agar is a creamy white powder soluble in hot water but insoluble in cold water.  

Agarose

Agarose is a neutral, long-chain polysaccharide formed by alternating D-galactose and 3,6-anhydro-alpha-L-galactopyranose residues joined by alpha-(1->3)- and beta-(1->4)-linkages. This electrically neutral polysaccharide is suitable for electrophoresis and chromatography. 

Uses

Agar is widely used in many industries due to its ability to form a gel. The large difference between gel-forming and melting temperatures gives agar its unique properties. 

1. Bacteriological agar is an indispensable ingredient in diagnostic labs and research projects, e.g., culture and AST, tissue culture, cell assays, etc. Due to the ease with which agar can be transported (dry, dissolved, and gelled), it is ubiquitous in the modern-day laboratory.  In the Microbiology lab, agar is the most commonly used growth medium for microorganisms. Agar media is essential for isolating and identifying microorganisms/pathogens from various samples. 
2. Agar is one of the most common basic media for gel electrophoresis, gel bead chromatography, and size exclusion chromatography. Due to its porous 3D framework, agar is frequently used in biomolecular separation and purification.
3. Agar has been fabricated in different forms (e.g., microspheres and films) to encapsulate molecules for sustained-drug delivery or immobilize proteins for tissue engineering. Due to the gelation property of agar, it is most often used as a hydrogel. Other applications of Agar are; emulsifier, carrier, lubricant, stabilizer, and laxative disintegrant in the pharmaceutical and cosmetic industries.
4. Applications in Food Industries
   1. Refined grades of agar are used in food applications, and agar is easier to use in food gels than many other substances. Common food applications of agar include puddings, custards, and soft candies. In Asian countries, jellies made from agar and natural fruit juices are very popular. Agar improves the texture of processed cheese and frozen desserts. 
   2. In the Bakery industry, agar is used in icings and frostings because it is compatible with large amounts of sugar. Its products neither melt at high storage temperatures nor stick to the packaging material. 
   3. Agar-agar serves as a preservative in food processing and is used in baked goods to inhibit staling. 
   4. Agar is also used for the preparation of canned meat and fish products. It is also used in retorted meat products such as canned corned beef.
   5. Agar-agar is high in dietary fiber (80 g per 100 g). Agar is used in low-calorie dishes, as it can not be digested in the gastrointestinal tract.  
   6. Agar’s properties are similar to gelatin. It is a good substitute for animal-based gelatin in vegetarian foods. 
   7. Agar is useful for the fermentation process.
5. In the agricultural industry, agar is a neutral carrier for nutrients and growth substances. Seedling germination herbaceous plants from meristematic tissue use agar as a nutrient substance.
6. Other uses of agar are; its usage in photographic emulsion, fermentation process, and making dental impressions, etc. 

Alternatives 

Apart from agar, media can be solidified by incorporating a gelling agent such as gelatin. Researches are underway to find newer and cost-effective alternatives to bacteriological agar. 

Some possible candidates are;  low-cost food-grade agar, cellulose produced by engineered bacteria, and fewer alternative gelling agents.

References and further readings 

1. Agar. Food and Agriculture Organization of the United Nations. Retrieved from https://www.fao.org/3/y4765e/y4765e06.htm on 1st November 2022.
2. Production, Properties and Uses of Agar. Food and Agriculture Organization of the United Nations. Retrieved from https://www.fao.org/3/x5822e/x5822e03.htm on 1st November 2022.
3. Vanesa Ayala-Nunez. #FEMSmicroBlog: Got agar? Say thanks to Angelina Hesse!Federation of European Microbiological Societies. Retrieved from https://fems-microbiology.org/femsmicroblog-got-agar-say-thanks-to-angelina-hesse/ on 1st November 2022.

Culture media are fundamental in diagnostic microbiology. Nearly all media are commercially available as ready-to-use agar plates or tubes of broth. Laboratories in high-income countries procure ready-to-use culture media from specialized manufacturers. 

Culture media can be prepared in-house by dissolving a specified amount of dehydrated media powder in distilled or deionized water, following the manufacturer’s recommendations. Laboratories in low and middle-income countries prefer preparing needed supplies of agars and broths using media powder instead of purchasing ready-to-use media. 

In-house culture media preparation has inherent challenges, so this blog post tries to tackle this issue by compiling best practices for media preparation. 

Manual preparation of culture media is labor intensive, so many companies have developed automated culture media preparation systems. If your lab can afford to purchase them, automated culture media preparation systems can save you manual labor and time.

Read more: Automated Culture Media Preparation and Dispenser System.

Selection of Media Types and Manufacturer 

When selecting culture media, look for products from reputable manufacturers supported by your national reference laboratory. Carefully check the product’s name, compositions, instructions for use, and shelf life. Look for humidity-proof packaging (screw cap, peel-off seal).

All bacteriology laboratories may need a minimal supply of basic nutrient media (such as nutrient agar or tryptic soy agar), enriched media (such as blood agar and chocolate agar), and selective media for Gram-positive (Columbia CNA agar), and Gram-negative bacteria (MacConkey agar). 

There is a plethora of media with similar applications. Depending on the setting, you may need to keep stock of a few media for epidemic preparedness (e.g., TCBS  in cholera risk settings). Get help from a clinical microbiologist to select the appropriate culture media needed for your laboratory. 

Procurement, Storage, and Stock Management 

Ensure your suppliers (both primary and backup) are reliable and provide after-sales support. Up on reception of goods, check and record;

1. product name and lot number
2. quantity received 
3. expiry date and remaining shelf life 
4. package integrity
5. order processing time
6. correctness of order delivery

At all times, follow the manufacturer’s instructions about storage conditions. For example, growth supplements such as egg yolk or antibiotic solutions should be stored at 2-8°C, whereas the base medium may be stored at room temperature, not exceeding 30°C.

Dehydrated media are stored in a cool, dry place, protected from light and dust. Monitor the storage area’s temperature and humidity. Do not store dehydrated media in the same room used for steam sterilizing, boiling materials, cleaning glassware, etc.

Organize the stock according to a first-in, first-out system, rank media in a logical order and separate opened from closed containers. 

The shelf life of dehydrated powder is usually several years. However, dehydrated media are hygroscopic (i.e., it absorbs water) and quickly deteriorate when exposed to moisture. When exposed to moisture, a hard mass is formed, which alters the chemical and microbiological properties of the medium. This can be a serious problem for tropical countries with humid climates.

Therefore, record the opening date on the container and use the product for a maximum of 6 months after opening unless otherwise specified by the manufacturer. After this period, visual and performance checks are required.  When not in use, keep containers tightly closed and seal the caps with adhesive tape. 

Discard products when expired, when the powder is clumped, discolored, or not free flowing, or in case of quality issues; record product details and reasons for discarding.

Preparation of Culture Media

Materials/Equipment Requirement

1. Pyrex flask (conical flask) at least double the size of the batch
2. Magnetic Stir Bar
3. Aluminum Foil
4. Weighing machine
5. Dehydrated culture media
6. Spatula
7. Graduated cylinder
8. Distilled or deionized water
9. Hot plate stirrer
10. Autoclave
11. Sterile Petri plates, and
12. Infrared no-touch thermometer

Calculation and Weighing

The dehydrated media should be calculated and weighed in a damp-free environment (low humidity), preferably under a laboratory fume hood. Ensure table, balance, and materials are clean, dust-free, and powder-free. 

Balance

Top loading (precision) balance is suitable for weighing media. Position the balance on a level, stable, vibration-free table. Calibrate the balance at regular intervals (e.g., annually) and verify control weights before use.

Materials and PPE

To prevent the risk of inhaling fine particles of dehydrated media, wear a dust mask while handling dehydrated media. Wear gloves to avoid skin contact. Wear safety goggles (e.g., for sodium deoxycholate) while using media with components that can cause skin and eye irritation (read product instructions). Wash hands before and after working with dehydrated culture media. 

Calculate

Follow the manufacturer’s instructions. Prepare a table with pre-defined weights and volumes for a fixed number of plates. To prepare a solid medium with a depth of 4 mm in a Petri plate of 9 cm diameter, a volume of 20-25 mL is needed. Record product name, lot number, preparation date, weight and volume of water, and operator identification in a logbook. 

Weighing

To facilitate weighing, use materials with pre-defined volume. Open the container and take the required amount of powder. Close the container immediately to protect it from humidity. Do not put the excess powder back into the bottle.

Mixing with Water and Heating

Boiling is often required to dissolve media powder, but specific manufacturers’ instructions printed in media package inserts should be followed exactly.  Use fresh water prepared by distillation, deionization, or reverse osmosis. The presence of copper ions, high conductivity, and high pH may significantly alter the quality of in-house prepared media. Do not use tap water as it affects selectivity and pH. 

The quality of glassware used for pouring media is also an important factor. Use Erlenmeyers, bottles, and graduated cylinders made of borosilicate glass to measure water and mix with media powder. If using reusable glassware, only borosilicate glassware should be used because soda glass can leach alkali into the media and change the pH of the medium, which may affect growth.

Procedure for dissolving the powder in water

1. Pour half of the required water into the flask, and add the pre-weighed powder. Pre-heating the water to 50-60°C  may facilitate dissolution. 
2. Stir or rotate for a few minutes (do not shake!)
3. Pour the rest of the water down the sides to dissolve any excess powder sticking to the flask walls (dry powder may not be sterilized in the autoclave)
4. Heating is required for dissolving agar-containing media. Do not close the flasks tightly.
5. Heat up to boiling, with frequent stirring, until the solution becomes clear. 
6. Avoid boiling, overheating and foaming, scorching and burning, clumping, and inconsistent mixing. 

Sterilization

Most media require sterilization, so only bacteria from patient specimens will grow and not contaminants from water or powdered media. Some media cannot be autoclaved (e.g., SS agar, Cary Blair agar) 

Liquid media are distributed to individual tubes or bottles before sterilization. Autoclave a medium only when the ingredients are completely dissolved. Do not tighten the lids or caps completely. Agar media are sterilized in large flasks or bottles capped with either plastic screw caps or plugs before being placed in an autoclave.

The timing of autoclave sterilization should start from the moment the temperature reaches 121°C and usually requires a minimum of 15 minutes.

The volume of the media in one sterilization batch should be kept small; ideally, only two liters should be autoclaved at a time. Sterilization indicators such as the Bowie Dick test and biological indicators such as spores of Bacillus stearothermophilus should be used to monitor the proper working of an autoclave.

Once the sterilization cycle is completed, molten agar is allowed to cool to approximately 50°C before being distributed to individual Petri plates (approximately 20 to 25 mL of molten agar per plate). 

Avoid

• Over-sterilization causes precipitation, pH change, component destruction 
• Under-sterilization results in contaminated medium

Ensure

• The wrapping allows for steam penetration
• There is enough space between the items to allow steam circulation
• To identify sterilized items use indicator tape to label the flask (medium name/code, preparation date, initials). 
• Use chemical indicators with each cycle (e.g., time-steam-temperature strips) and biological indicators on an interval basis to verify autoclave cycles. 

Note

• Use a ‘cool down program’ or wait until the pressure drops sufficiently before opening (70-80°C) to avoid fast pressure drop (liquid can boil over, and caps can be blown off). 
• Do not wait too long before unloading the autoclave; overheating may destroy media ingredients. 

Fine-tuning (supplements and pH)

If other ingredients are to be added (e.g., supplements such as sheep blood or specific vitamins, nutrients, growth promoters, or antibiotics), they should be incorporated when the molten agar has cooled, just before distribution to plates.

The quality of the blood plays an important role in the performance of the blood-containing media, e.g., hemolytic reactions are well distinguished in sheep blood-containing media. The blood’s concentration, homogeneity, viscosity, and color should be checked before it is used for media preparation. The certificate of analysis and sterility conditions should be considered for other additives.

Delicate media components that cannot withstand steam sterilization by autoclaving (e.g., serum, certain carbohydrate solutions, certain antibiotics, and other heat-labile substances) should be sterilized separately by membrane filtration. Passage of solutions through membrane filters with pores ranging in size from 0.2 to 0.45 um in diameter will not remove viruses but does effectively remove most bacterial and fungal contaminants. 

Dispensing of Prepared Culture Media 

Cool down the culture media in a water bath (45-50°C) or hot plate stirrer before dispensing to minimize condensation. You can check the temperature with an infrared no-touch thermometer. Dispensing at too high temperature leads to excessive evaporation. If media stay too long in a water bath, it may cause precipitation (reheat, but do not overheat). Do not use cold water to cool down agar media, as it may lead to flakes or cloud formation. 

If using reusable glass Petri plates, sterilize them at 160°C for 2 hours in a hot air oven. Allow the oven to cool to 50°C before opening (to avoid cracking glassware). Dispense the media aseptically in a draught-free room (with closed windows), and avoid fans or climate control.  Work close to the flame or in a biological safety cabinet

Procedure for dispensing agar media in plates

1. Flame sterilize the neck of the flask before and between pouring
2. Mix the culture media gently by rotating the flask before dispensing
3. Dispense on a level surface
4. For antimicrobial susceptibility testing, agar depth should be 4 ± 0.5 mm: Circular Petri plate of 90 mm: about 25 mL
5. Use sterile, graduated pipettes or media distribution syringe/pump
6. Avoid forming air bubbles (flame surface or use a heated loop to remove them). 

Dispensing of agar or liquid media in tubes

1. Use tubes with lids that allow ventilation (e.g., screw caps), do not tighten completely. 
2. Put tubes in an autoclavable rack.
3. Mix gently by rotating the flask before dispensing
4. Dispense the correct volume per tube. Use sterile, graduated pipettes or a media distribution syringe/pump
5. Close screw caps tightly after autoclaving

Drying of plates/tubes

1. Dry for several hours at room temperature (up to 24 hours) to remove condensation. 
2. Selective media: ± 30 minutes with lid ajar. If contamination risk: keep lids closed. 
3. Dry before packing to prevent condensation on the lids. Avoid over-drying (cracks). 
4. Drying depends on the type of media:
   1. For agar slants, let dry in a sloped position to give a butt of 2.5-3 cm deep and a slope of 2-2.5 cm long. Use a standardized and validated rack. 
   2. For agar, semi-solid tubes, and liquid media: let dry in a rack (vertical position).

Packing and Storage

Label individual plates with name (abbreviated/code), preparation date, and lot number. Wrap the plates in sealed, labeled plastic bags, a maximum of ten plates per bag, to avoid moisture. Store them upside down, at 2-8°C, in the dark following manufacturer’s instructions.

Shelf life

Agar media in Petri plates. 

• Blood agar: 7 days; with unstable additives: 2-5 days
• Most selective media: 5-7 days
• Nutrient agar without blood: 2-4 weeks

In tubes 

• Simple, non-selective broths and agars: 6 months
• Selective media: 3 weeks (2-8 weeks)
• Selenite broth: 2-3 months

Quality Control

Finally, all media, whether purchased or prepared, must be subjected to stringent quality control before being used in the diagnostic setting. Quality control (QC) should be based on a pragmatic, risk-based approach. Newly prepared and dispensed media should be quarantined until they pass QC. Read more about quality control of culture media. 

Using Culture media

Bring the culture media to room temperature before use. Ensure there are no visible drops of water on the agar surface or inside the lid. If seen, do not shake off condensation water from the lid. 

Instead, dry plates for 20-30 minutes at 35-37°C with agar plates upside down and agar base resting at an angle on the lid (if necessary, dry plates at 20-25°C overnight). 

Do not over-dry plates (cracks in surface, surface wrinkled). 

Visual sterility check before use is a mandatory step. Check the plates for contamination or growth of colonies. 

Disposal of used culture media

For disposal of used culture media, follow in-country or WHO guidelines, which recommend inactivation and subsequent incineration before disposal. Inactivation (so-called ‘destruction’ or Level III inactivation) should be done in the laboratory, preferably by autoclaving. 

Most Common Errors and Possible Causes 

Clumping of dehydrated culture media

• Humidity too high during storage
• The container was left open for too long or was not closed tightly after opening 
• Dehydrated culture medium beyond the shelf life

Wrong pH

• pH meter not calibrated 
• pH verification done on too hot medium (generally to be done at 25°C) 
• Use of poor-quality water or container 
• Use of chemically contaminated containers Incomplete dissolution/mixture of medium
• Dehydrated medium stored incorrectly (e.g., not tightly closed) or beyond the shelf life

Incomplete solubility

• Use of inadequate water
• Inadequate heating/inadequate timing for dissolution 
• Insufficient soaking or incomplete mixing 
• Flask too small to allow adequate mixing and/or convection

Darkening, caramelization

• Overheating: excessive sterilization, heterogeneous mixing medium kept at 50°C for too long, repeated re-melting or too high temperature. 
• Incomplete dissolution of medium

Incomplete gelling or soft agar

• Incorrect proportions of product to water: error in weighing or over-dilution
• Agar not properly dissolved: poor mixing prolonged storage at 50°C
• Overheating of culture medium, possibly at low pH
• Repeated re-melting causes overheating

Turbidity, precipitation

• Poor quality of dehydrated media
• Use of poor-quality of water or container
• Overheating: excessive sterilization, heterogeneous mixing medium kept at 50°C for too long, repeated re-melting or too high temperature. 
• Wrong pH 
• Incomplete dissolution/mixture of medium 
• Loss of water of the prepared culture medium due to evaporation 

Poor growth or loss of differential properties 

• Incorrect or improperly maintained QC organisms used 
• Overheating: excessive sterilization, heterogeneous mixing medium kept at 50°C for too long, repeated
• re-melting or at too high temperature
• Incomplete dissolution/mixture of medium
• Inhibitory substances in water, container or inoculum Wrong PH

References and further readings

1. Bailey & Scott’s Diagnostic Microbiology, Forbes, 11th edition
2. Orekan J, Barbé B, Oeng S, Ronat JB, Letchford J, Jacobs J, Affolabi D, Hardy L. Culture media for clinical bacteriology in low- and middle-income countries: challenges, best practices for preparation and recommendations for improved access. Clin Microbiol Infect. 2021 Oct;27(10):1400-1408. doi: 10.1016/j.cmi.2021.05.016. Epub 2021 May 18. PMID: 34015533.