'''Granada medium''' is a selective and differential culture medium designed to selectively isolate ''Streptococcus agalactiae'' (Group B streptococcus, GBS) and differentiate it from other microorganisms. Granada Medium was developed by Manuel Rosa-Fraile et al. at the Service of Microbiology in the Hospital Virgen de las Nieves in Granada (Spain).<ref name="Rosa 1992" />

Identification of GBS on granada medium is straightforward and relies on detection of granadaene, a red polyenic pigment specific of GBS.<ref name="Rosa-Fraile 1999">{{cite journal|vauthors=Rosa-Fraile M, Rodriguez-Granger J, Cueto-Lopez M, Sampedro A, Biel Gaye E, Haro M , Andreu A|title=Use of Granada Medium To Detect Group B Streptococcal Colonization in Pregnant Women|journal = Journal of Clinical Microbiology|date=1999|volume=37|issue=8|pages=2674–2677|doi=10.1128/JCM.37.8.2674-2677.1999|pmid=10405420|pmc=85311}}</ref><ref name="Rosa-Fraile 2006">{{cite journal|vauthors=Rosa-Fraile M, Rodriguez-Granger J, Haidour-Benamin A, Cuerva JM, Sampedro A|title=Granadaene: Proposed Structure of the Group B Streptococcus Polyenic Pigment|journal = Applied and Environmental Microbiology|date=2006|volume=72|issue=9|pages=6367–6370|doi=10.1128/aem.00756-06|pmid=16957264|pmc=1563658|bibcode=2006ApEnM..72.6367R}}</ref><ref name="Rosa-Fraile 2017">{{cite journal|vauthors=Rosa-Fraile M, Spellerberg B|title=Reliable Detection of Group B Streptococcus in the Clinical Laboratory.|journal = Journal of Clinical Microbiology|date=September 2017|volume=55|issue=9|pages=2590–2598|doi=10.1128/JCM.00582-17|pmid=28659318|pmc=5648696}}</ref>

thumb|''Streptococcus agalactiae'' on granada agar, anaerobic incubation thumb|''Streptococcus agalactiae'' on granada broth

Granada medium is marketed in the US by Hardy Diagnostics<ref name="Hardy">{{cite web |title=Granada Medium |url=https://hardydiagnostics.com/media/assets/product/documents/GranadaMedium.pdf |website=Haerdy Diagnostics |access-date=21 October 2023}}</ref> and in the European Union and UK as a trade mark (®) by Biomerieux<ref name="Biomerieux">{{cite web |title=Granada agar |url=https://www.biomerieux-culturemedia.com/product/12-granada-agar# |website=Biomerieux |access-date=21 October 2023}}</ref> and Becton Dickinson.<ref name="BD">{{cite web |title=BD Group B Streptococcus Differential Agar (Granada Medium) |url=https://www.bd.com/resource.aspx?idx=13200 |access-date=21 October 2023}}</ref>

== Composition== {| class="wikitable" |- ! Ingredient<ref name="Rosa 1992">{{cite journal|vauthors=Rosa M, Perez M, Carazo C, Peis JI, Pareja L, Hernandez F|title=New Granada Medium for Detection and Identification of Group B Streptococci|journal = Journal of Clinical Microbiology|date=1992|volume=30|issue=4|pages=1019–1021|doi=10.1128/JCM.30.4.1019-1021.1992| pmc=265207|pmid=1572958}}</ref> !! Amount!! Function |- | Agar|| 10g || Gelling agent |- | Bacto™ Proteose Peptone #3, (Difco) BD || 25g || Specific nutrient, It cannot be substituted by any alternative peptone |- | Starch || 20g || Pigment stabilizer |- | Glucose || 2.5g || Nutrient |- | Horse serum || 15ml || Nutrient |- | MOPS ( 3-(N-morpholino)propanesulfonic acid) hemisodium salt || 11g || Good's buffer |- | Disodium hydrogen phosphate || 8.5g || Buffer |- | Sodium pyruvate || 1g || Additional source of energy, protective effects against reactive oxygen species |- | Magnesium sulfate || 0.2g || ------ |- | Methotrexate || 6&nbsp;mg || Pigment enhancer |- | Crystal violet || 0.2&nbsp;mg || Inhibit the growth of gram-positive bacteria |- | Colistin sulfate || 5&nbsp;mg || Inhibit the growth of gram-negative bacteria |- | Metronidazole || 1&nbsp;mg || Inhibit the growth of anaerobic bacteria |- | Water|| 1000ml || -------|

|}

pH 7.45±0.1

== Background and principles == Granada medium was developed for selective isolation and identification of GBS from clinical specimens.<ref name="Rosa 1992" /> Production of a red pigment (granadaene) on granada medium is unique to β-hemolytic group B streptococci isolated from humans.<ref name="Rosa-Fraile 2014">{{cite journal|vauthors=Rosa-Fraile M, Dramsi S, Spellerberg B|title=Group B streptococcal haemolysin and pigment, a tale of twins|journal = FEMS Microbiology Reviews|date=2014|volume=38|issue=5|pages=932–946|doi=10.1111/1574-6976.12071|pmid=24617549 |pmc=4315905 }}</ref>

Granadaene is a non-isoprenoid polyenic pigment (ornithinrhamnododecaene) with a conjugated system of 12 double bonds.<ref name="Rosa-Fraile 2006" /><ref name="Paradas 2012">{{cite journal|vauthors=Paradas M, Jurado R, Haidour A, Rodríguez Granger J, Sampedro Martínez A, de la Rosa Fraile M, Robles R, Justicia J, Cuerva JM |title=Clarifying the structure of granadaene: total synthesis of related analogue [2]-granadaene and confirmation of its absolute stereochemistry.|journal = Bioorganic & Medicinal Chemistry|date=2012|volume=20|issue=22|pages=6655–6651|doi=10.1016/j.bmc.2012.09.017|pmid=23043725}}</ref><ref name="Madden 2014">{{cite journal|vauthors=Madden KS, Mosa FA, Whiting A|title=Non-isoprenoid polyene natural products – structures and synthetic strategies|journal = Organic and Biomolecular Chemistry|date=2014|volume=12|issue=40|pages=7877–7899|doi=10.1039/C4OB01337A|pmid=25188767|url=https://durham-repository.worktribe.com/output/1418047 }}</ref>

β-hemolysis and pigment production are encoded in GBS by a gene cluster of 12 genes, the ''cyl'' cluster.<ref name="Spellerberg 1999">{{cite journal|vauthors=Spellerberg B, Pohl B, Haase G, Martin S, Weber-Heynemann J, Lutticken R|title=Identification of Genetic Determinants for the Hemolytic Activity of Streptococcus agalactiae by ISS1 Transposition.|date=1999|volume=181|issue=10|pages=3212–3219|pmc=93778|pmid=10322024|journal = Journal of Bacteriology|doi=10.1128/JB.181.10.3212-3219.1999}}</ref><ref name="Spellerberg 2000">{{cite journal|vauthors=Spellerberg B, Martin S, Brandt C, Lutticken R|title=The cyl genes of Streptococcus agalactiae are involved in the production of pigment.|journal = FEMS Microbiology Letters|date=2000|volume=188|issue=2|pages=125–128|doi=10.1016/s0378-1097(00)00224-x|pmid=10913694|doi-access=free}}</ref> Moreover, it has been suggested that GBS pigment and hemolysin are identical or closely related molecules and it has also been reported that they are important factors contributing to GBS virulence.<ref name="Rosa-Fraile 2014" /><ref name="Whidbey 2013">{{cite journal|vauthors=Whidbey C, Harrell MI, Burnside K, Ngo L, Becraft AK, Iyer LM, Aravind L, Hitti J, Adams Waldorf KM, Rajagopal L|title=A hemolytic pigment of Group B Streptococcus allows bacterial penetration of human placenta.|journal = Journal of Experimental Medicine|date=2013|volume=219|issue=6|pages=1265–1281|pmc=3674703|pmid=23712433|doi=10.1084/jem.20122753}}</ref><ref name=Whidbey2015>{{cite journal|vauthors=Whidbey C, Vornhagen J, Gendrin C, Boldenow E, Samson JM, Doering K, Ngo L, Ezekwe EA Jr, Gundlach JH, Elovitz MA, Liggitt D, Duncan JA, Adams Waldorf KM, Rajagopal L|title=A streptococcal lipid toxin induces membrane permeabilization and pyroptosis leading to fetal injury.|journal = EMBO Molecular Medicine|date=2015|volume=7|issue=4|pages=488–505|pmc=4403049|pmid=25750210|doi=10.15252/emmm.201404883 }}</ref>

=== Components === Granada agar consists primarily of a proteose peptone starch agar buffered with MOPS (a Good's buffer) and phosphate and supplemented with methotrexate and antibiotics.<ref name="Rosa 1992" /> Proteose peptone, horse serum, glucose and sodium pyruvate provide nutrients for the growth of ''Streptococcus agalactiae'', sodium pyruvate provide also protective effect against reactive oxygen species (ROS). MOPS and phosphate buffer the medium. Methotrexate triggers pigment production<ref name="Rosa-Fraile 2014" /> and starch stabilizes the pigment.<ref name="Rosa-Fraile 2014" /> The selective supplement contains the antibiotics, colistin (inhibitory for gram-negative bacteria) and metronidazole (inhibitory for anaerobic bacteria), and crystal violet to suppress the accompanying gram-positive bacteria. thumb|Granadaene

A key component of granada medium is Proteose Peptone N3 (Difco & BD). This pepsic peptone was developed by DIFCO (Digestive Ferments Company) during the First World War for producing bacterial toxins for vaccine production.<ref name="Difco & BBL Manuel">{{cite book|title=Difco & BBL. Manual of Microbiological Culture Media 2nd Edition|publisher=BD Diagnostics – Diagnostic Systems|isbn=978-0-9727207-1-7|year=2009|page=450}}</ref>

Fort development of red-brick colonies of GBS in granada medium it is necessary the presence of the peptide Ile-Ala-Arg-Arg-His-Pro-Tyr-Phe in the culture medium. This peptide only is produced during the hydrolysis with pepsin of mammal albumin.<ref name="Rosa-Fraile 1999-Peptide">{{cite journal|vauthors=Rosa-Fraile M, Sampedro A, Varela J, Garcia-Peña M, Gimenez-Gallego G |title=Identification of a peptide from mammal albumins responsible for enhanced pigment production by group B streptococci.|journal = Clin Diagn Lab Immunol|date=1999|volume=6|issue=3|pages=425–426|doi=10.1128/CDLI.6.3.425-426.1999|pmid=10225848|pmc=103735}}</ref>

For optimal production of pigment it is also necessary the presence in the peptone of other substances (uncharacterized at present) from mammal gastrointestinal wall tissues used to prepare some peptones.<ref name="Camacho 2005">{{cite book|last1=Enrique Camacho Muñoz|title=Importancia de la Proteosa Peptona No 3 en la Producción de Pigmento por ''Streptococcus agalactiae'' en el Medio Granada|date=2005|publisher=Universidad de Granada|isbn=978-84-338-3741-7|url=http://digibug.ugr.es/bitstream/10481/853/1/15843786.pdf|access-date=24 June 2016}}</ref>

The presence of starch is a basic requirement to stabilize the pigment allowing the development of red colonies of GBS.<ref name="Rosa-Fraile 2014" />

Nevertheless, if soluble starch is used it results in a culture medium that deteriorates quickly at room temperature because soluble starch is hydrolysed by serum (added as supplement) amylase. This drawback can be addressed either not using serum or using unmodified starches to prepare the culture medium, because unmodified starches are more resistant to the hydrolytic action of amylase.<ref name="Rosa-Fraile 2005">{{cite journal|vauthors=Rosa-Fraile M, Rodríguez-Granger J, Camacho-Muñoz E, Sampedro A |title=Use of unmodified starches and partial removal of serum to improve Granada medium stability.|journal = Journal of Clinical Microbiology|date=2005|volume=43|issue=4|pages=18889–1991|doi=10.1128/JCM.43.4.1989-1991.2005|pmid=15815040|pmc=1081375}}</ref>

== Uses == GBS grows on granada agar as pink-red colonies after 18–48 hours of incubation (35–37&nbsp;°C), better results are obtained in anaerobiosis (culturing in an anaerobic environment).<ref name="Rosa 1992" />

Granada agar is used for the primary isolation, identification and screening of β-hemolytic GBS from clinical specimens.<ref name="Rosa-Fraile 1999" /><ref name="Rosa-Fraile 2017" /> This culture medium is selective for GBS, nevertheless other microorganisms (such as enterococci and yeasts), resistant to the selective agents used, can develop as colorless or white colonies.<ref name="Rosa-Fraile 1999" />

thumb|Red colonies of ''Streptococcus agalactiae'' on granada agar. Vagino-rectal culture 18h incubation 36°C anaerobiosis

Granada agar is useful for the screening of pregnant women for the detection of vaginal and rectal colonization with GBS to use intrapartum antibiotic prophylaxis to avoid early-onset GBS infection in the newborn.<ref name="Rosa-Spelleerberg 2017">{{cite journal |last1=Rosa-Fraile M, Spellerberg B. |title=Reliable Detection of Group B Streptococcus in the Clinical Laboratory. |journal=J Clin Microbiol |date=2017 |volume=55 |issue=9 |pages=2590–2598 | doi=10.1128/JCM.00582-17 |pmid=28659318 |pmc=5648696 }}</ref><ref name="Verani 2010">{{cite journal|vauthors=Verani JR, McGee L, Schrag SJ| title= Prevention of Perinatal Group B Streptococcal Disease Revised Guidelines from CDC, 2010.|journal =MMWR Recomm Rep |date=2010|volume=59|issue=RR-10|pages=1–32| pmid= 21088663|url=https://www.cdc.gov/mmwr/pdf/rr/rr5910.pdf}}</ref> <ref name="Filkins 2020 ASM">{{cite journal |last1=Laura Filkins, Jocelyn R Hauser, Barbara Robinson-Dunn, Robert Tibbetts, Bobby L Boyanton Jr, Paula Revell, American Society for Microbiology Clinical and Public Health Microbiology Committee, Subcommittee on Laboratory Practices |title=American Society for Microbiology provides 2020 Guidelines for Detection and Identification of Group B Streptococcus |journal = Journal of Clinical Microbiology|date=2020 |volume=JCM.01230-20. |issue=1 |pages=e01230-20 |doi=10.1128/JCM.01230-20|pmc=7771461 |pmid=33115849 |s2cid=226049927 |url=https://jcm.asm.org/content/jcm/early/2020/10/26/JCM.01230-20.full.pdf |access-date=14 December 2020|doi-access=free }}</ref><ref name="ASM 2020">{{cite web |last1=Filkins L, Hauser J, Robinson-Dunn B, Tibbetts R, Boyanton B, Revell P. |title=Guidelines for the Detection and Identification of Group B Streptococcus. American Society for Microbiology. 2020 |url= https://asm.org/Guideline/Guidelines-for-the-Detection-and-Identification-of }}</ref> It has also been suggested that GBS pigmentation on Granada agar can help to identify pregnant women and newborns at increased risk for developing invasive GBS disease<ref name="Huebner 2022">{{cite journal |last1=Huebner EM, Gudjónsdóttir MJ, Dacanay MB, Nguyen S, Brokaw A, Sharma K, Elfvin A, Hentz E, Rivera YR, Burd N, Shivakumar M, Coler B, Li M, Li A, Munson J, Orvis A, Coleman M, Jacobsson B, Rajagopal L, Adams Waldorf KM. |title=Virulence, phenotype and genotype characteristics of invasive group B Streptococcus isolates obtained from Swedish pregnant women and neonates |journal=Annals of Clinical Microbiology and Antimicrobials |date=2022 |volume=21 |issue=1 |page=43 |doi=10.1186/s12941-022-00534-2 |pmid=36229877 |pmc=9560721 |doi-access=free }}</ref>

== Procedure == The specimens can be directly streaked on a plate of granada agar or after an enrichment step to obtain maximum isolation.<ref name="Verani 2010" /> Specimens should be streaked as soon as possible after they are received in the laboratory. If material is being cultured from a swab (e.g.- from a vaginal or vagino-rectal swab), roll swab directly onto the agar plate to provide adequate exposure of the swab to the medium for maximum transfer of organisms. Place the culture in an anaerobic environment, incubate at 35-37&nbsp;°C, and examine after overnight incubation, and again after approximately 48 hours.<ref name="Rosa 1992" />

To increase recovery of GBS, swabs can also be inoculated previously into a selective enrichment broth medium, such as the Todd-Hewitt broth supplemented with gentamicin or colistin and nalidixic acid and incubated for 18–24 hours at 35-37&nbsp;°C.<ref name="Verani 2010" /><ref name="Filkins 2020 ASM">{{cite journal |last1=Laura Filkins, Jocelyn R Hauser, Barbara Robinson-Dunn, Robert Tibbetts, Bobby L Boyanton Jr, Paula Revell, American Society for Microbiology Clinical and Public Health Microbiology Committee, Subcommittee on Laboratory Practices |title=American Society for Microbiology provides 2020 Guidelines for Detection and Identification of Group B Streptococcus |journal = Journal of Clinical Microbiology|date=2020 |volume=JCM.01230-20. |issue=1 |pages=e01230-20 |doi=10.1128/JCM.01230-20|pmc=7771461 |pmid=33115849 |s2cid=226049927 |url=https://jcm.asm.org/content/jcm/early/2020/10/26/JCM.01230-20.full.pdf |access-date=14 December 2020|doi-access=free }}</ref><ref name="ASM 2020" />

== Results == Colonies of β-hemolytic GBS appear on granada medium as pink or red colonies, and they are easily distinguished from other microorganisms that may have also grown on the plate. Any degree of orange development should be considered indicative of a GBS colony, and further identification tests are not necessary.<ref name="Rosa-Fraile 1999" /> Non-β-hemolytic GBS develops on granada agar as white colonies that, if necessary, can be further tested using latex agglutination or the CAMP test.<ref name="Rosa-Fraile 2017"/><ref name="Verani 2010" />

== Variant == thumb|Colonies of ''Streptococcus agalactiae'' on granada agar, aerobiosis, coverslip technique Granada agar plates can also be incubated aerobically provided that a coverslip is placed over the inoculum on the plate.<ref name="Rosa-Fraile 1999" />

Granada medium can also be used as liquid media (granada broths)<ref name="Carvalho">{{cite journal |last1=Carvalho Mda G, Facklam R, Jackson D, Beall B, McGee L. |title=Evaluation of three commercial broth media for pigment detection and identification of a group B Streptococcus (Streptococcus agalactiae). |journal=J Clin Microbiol |date=2009 |volume=47 |issue=12 |pages=4161–4163 |doi=10.1128/JCM.01374-09 |pmid=19812277 |pmc=2786674 }}</ref> such as Strep B carrot broth<ref name="Carrot">{{cite web |title=STREP B CARROT BROTH™ |url=https://hardydiagnostics.com/media/assets/product/documents/StrepBCarrotBrothOneStep.pdf|access-date=17 October 2023}}</ref> When using granada media liquids anaerobic incubation is not necessary.<ref name="Rosa-Fraile 1999" />

== Granadaene and ''Streptococcus agalactiae'' == β-hemolysis and pigment (granadaene) production are encoded in GBS by a gene cluster of 12 genes, the ''cyl'' cluster.<ref name="Spellerberg 1999" /><ref name="Spellerberg 2000" />

Moreover, it has been suggested that GBS pigment and hemolysin are identical or closely related molecules and it has also been reported that they are important factors contributing to GBS virulence.<ref name="Rosa-Fraile 2014" /><ref name="Whidbey 2013" /><ref name=Whidbey2015 />

Nevertheless, 1–5% of GBS strains are non-hemolytic and do not produce pigment.<ref name="Rosa-Fraile 2014" /> However these non-hemolytic and non-pigmented GBS strains (lacking pigment and hemolysin) are considered less virulent. <ref name="Whidbey 2013" /><ref name=Whidbey2015 /><ref name="Whidbey Dissertation">{{cite book|last1=Christopher-Mychael Whidbey|title=Characterization of the Group B Streptococcus Hemolysin and its Role in Intrauterine Infection|date=2015|publisher=University of Washington|url=https://digital.lib.washington.edu/researchworks/bitstream/handle/1773/33228/Whidbey_washington_0250E_14193.pdf|access-date=23 June 2016}}</ref><ref name="Rodriguez-Granger 2015">{{cite journal|vauthors=Rodriguez-Granger J, Spellerberg B, Asam D, Rosa-Fraile M |title=Non-haemolytic and non-pigmented group b streptococcus, an infrequent cause of early onset neonatal sepsis.|journal = Pathogens and Disease|date=2015|volume=73|issue=9|doi=10.1093/femspd/ftv089|pmid=26449711|article-number=ftv089|pmc=4626576}}</ref><ref name="Armistead 2019">{{cite journal |last1=Armistead B, Oler E, Adams Waldorf K, Rajagopal L. |title=The Double Life of Group B Streptococcus: Asymptomatic Colonizer and Potent Pathogen |journal = Journal of Molecular Biology|date=2019 |volume=431 |issue=16 |pages=2914–2931 |doi=10.1016/j.jmb.2019.01.035 |pmid=30711542 |pmc=6646060 }}</ref><ref name="Armistead Quach 2020">{{cite journal |last1=Armistead B, Quach P, Snyder JM, Santana-Ufret V, Furuta A, Brokaw A, Rajagopal L. |title=Hemolytic membrane vesicles of Group B Streptococcus promote infection. |journal = The Journal of Infectious Diseases|date=2020 |volume=jiaa548 |issue=8 |pages=1488–1496 |doi=10.1093/infdis/jiaa548 |pmid=32861213 |pmc=8064051}}</ref>

== References == {{Reflist}}

Category:Microbiological media Category:Cell culture media