Microbiologylog: Salmonella spp. from Blood culture

Salmonella are predominantly motile (most species except Salmonella pullorum-gallinarum are motile with peritrichous flagella), non-spore forming, Gram negative rod bacteria that belongs to Enterobacteriaceae family. They are divided into two species – Salmonella bongori (previously serotype V) and Salmonella enterica (Salmonella enterica is subdivided into subspecies – enteric (serotype I), salamae (serotype II), arizonae (serotype IIIa), diarizonae (serotype IIIb), houtenae (serotype IV) and indica (serotype VI), and over 2500 serovars. The serovars are defined based on the somatic O (Lipopolysaccharide) and flagellar H antigens (The Kauffman-White classification).

The blood culture aerobic and anaerobic bottle flagged up positive after 8 hours incubation in the BD Bactec FX Blood culture analyser and upon Gram stain shows Gram negative rods. Routine blood culture plates where inoculated according the Laboratory SOP - Blood agar, chocolate agar, UriSelect chromogenic agar and FAA AN anaerobic plates. All culture plates were purchased from E & O Labs (http://www.eolabs.com/). After 18-48 hours incubation at appropriate temperatures and condition, there was growth on all plates. The Salmonella isolate on culture plates are shown below.

Culture medium	Incubation condition	Incubation Time	Appearance
Blood agar	CO₂at 37^OC	18 – 48 hours	moist, grey, round non-haemolytic colony
Chocolate agar	CO₂at 37^OC	18 – 48 hours	moist, grey, round colony
UriSelect chromogenic agar	Air at 37^OC	18 – 24 hours	moist, white/cream, round colony

This isolate was then identified using Biomerieux Vitek MS (MALDI-TOF) as Salmonella group. Purity plates were done on CLED plates and used for agglutination test after 24 hours incubation at the appropriate temperature condition. Salmonella isolates are used for serological confirmation using the agglutination test Polyvalent O and Polyvalent H specific antisera. This is an easy, quick and straightforward method. Specialist reference microbiology laboratories use more advanced and automated methods such as PCR technique.

The Kaufmann-White scheme is a system complex serotyping scheme that classifies Salmonella into serotypes based on their surface antigens. Specific antisera are raised against three antigenic sources; the ‘O’ polysaccharide component of the bacterial cell wall, the ‘H’ flagellar antigens present in most strains and the ‘Vi’ virulence/capsular antigen present mainly, but not exclusively, in S.typhi. It was further found that each strain could possess several ‘O’ determinants and so could be attributed to a particular ‘O’ group. Additional subdivision could also be made on the basis of flagella ‘H’ antigens as Salmonella exhibit phase variation between motile (phase 1) and non-motile (phase 2) phenotypes. The Kaufmann-White scheme was first published in 1934 listing 44 serotypes, but now contains serotypes for more than 2500 Salmonella spp and is used both in routine and specialised laboratories for the identification of Salmonella species. Examples of the serotypes can be seen on the table below

Serotypes	‘O’ Antigens	‘H’ Antigens
Serotypes	‘O’ Antigens	Phase 1	Phase 2
Salmonella typhi	9,12,Vi	d	None
Salmonella paratyphi A	1,2,12	a	None
Salmonella paratyphi A var. Durazzo	2,12	a	None
Salmonella paratyphi B	1,4,5,12	b	1,2
Salmonella paratyphi B var. Odense	1,4,12	b	1,2
Salmonella enteritidis	1,9,12	g,m	None
Salmonella typhimurium	1,4,5,12	i	1,2