A 2-year old male presented with fever and diarrhea. Stool culture identified Salmonella enterica serotype Heidelberg, matching an outbreak linked to chicken. This led to antibiotic treatment and recovery, altering the patient's care. Salmonella is classified into two species, with S. enterica having over 2500 serotypes commonly transmitted by food like chicken. Identification and antibiotic guidance helps manage high-risk cases and limit resistance.

1. Ian McHardy, Ph.D.
University of California, Los Angeles (UCLA)
San Franciscan Chicken

2. Patient History
• A 2-year old male with a very complicated medical history
presented to the UCLA emergency department with fever,
diarrhea, myalgias, rhinorrhea, dehydration, and decreased
activity.
• The patient had a medical history significant for heart
transplant and post-transplant lymphoproliferative disorder.
• He lived with 3 siblings; one of which was sick with similar
diarrheal symptoms.

3. Enteric Work-up at UCLA
Specimen: Fresh stool was collected and sent in a transport container to the UCLA clinical microbiology
lab for culture.
Workup in the clinical microbiology lab:
1. Stool plated onto:
– Blood agar plate (BAP)
– MacConkey’s agar plate
– Hektoen Enteric (HE) agar plate
– Campy Blood agar plate
2. Resulting colonies analyzed by:
– Colony morphology
– Biochemical tests
– Antibody-based serogrouping tests
3. Findings are reported to attending physicians.
Photo Credit: ASM Microbe Library (Hudzicki)

4. Stool work-up plates
Sheep Blood agar
• Contains blood and nutrients for growth of many
bacteria. Both Gram-positive and Gram-negative
bacteria can grow.
MacConkey agar –
• Contains bile salts and crystal violet, which inhibit the
growth of most Gram-positive bacteria. Bacteria that
ferment lactose appear pink on this agar.
Hektoen Enteric (HE) agar
• HE contains bile salts, lactose, acid fuchsin and bromo
thymol blue indicators. Lactose fermenters produce
yellow – orange colonies. Colonies that produce
hydrogen sulfide (H2S) appear black.
Photo Credit: ASM Microbe Library (Buxton) and ASM Microbe Library (Miller and Hanley), and ASM Microbe Library (Hudzicki).

5. API 20E by bioMerieux
Principle:
The strip consists of 20 microtubes containing dehydrated substrates. The tests are
inoculated with bacterial suspensions that reconstitutes the media. During incubation,
specific bacterial metabolites are produced that can be detected via color changes.
Based on this information, bacterial identification is often possible.
Photo Credit: Wikimedia

6. Wellcolex Serogrouping for Salmonella
Antibody conjugated latex agglutination test for rapid serogroup classification of Salmonella spp.
Latex reagent 1:
• Green latex (anti-Salmonella grp. D1 Abs).
• Blue latex (anti-Salmonella grp. C Abs).
• Red latex (anti-Salmonella grp. B Abs).
Latex reagent 2:
• Blue latex (anti-Salmonella grps. E & G Abs).
• Red latex (anti-Vi Abs).
• Green latex (anti-Salmonella grp. A Abs).
Photos Provided by UCLA.

7. UCLA Patient
Stool microbiology findings for the 2 year-old patient:
1. Black colonies grew on Hektoen-Enteric agar.
2. Biochemical tests on sub-cultured black colonies indicated the organism was a Salmonella species.
3. Wellcolex serogrouping determined the isolate was a Salmonella enterica subspecies enterica
serogroup B (Red latex from reagent 1).
4. Antimicrobial susceptibility testing was performed.
5. Los Angeles County Public Health Laboratory determined the isolate as a Salmonella enterica
subspecies enterica serotype Heidelberg (part of a larger US outbreak).
Antibiotic Sensitivity MIC in µg/mL
Ampicillin Resistant >32
Ciprofloxacin Susceptible 0.03
Bactrim Susceptible <=1/20

8. UCLA Patient
•Before culture completion, the patient was discharged without antibiotics for presumed viral enteritis.
•Given the patient’s age and complicated medical history, antibiotics were warranted to ensure full
recovery. Antibiotics were prescribed and the patient improved.
The clinical microbiologist provided an unexpected diagnosis that resulted in a dramatic
change in patient care.
Photo Credit: Centers for Disease Control and Prevention

9. Salmonella Heidelberg Outbreak, 2013
• 74% of the 389 infected persons were from California.
• Among 389 persons with available information, 40% reported being hospitalized.
• 14% of ill persons developed blood infections as a result of their illness.
• Typically, only ~5% of persons ill with Salmonella infections develop blood
infections.
• No deaths were reported.
• Several isolates were multi-drug resistant.
• At least some of the cases were attributable to
Foster Farms Chicken purchased at Costco in
San Francisco, CA.
Photo Credit: Centers for Disease Control and Prevention

10. Classification - Salmonella species
and serotypes
Only two species of the genus Salmonella are recognized:
• S. enterica – composed of six sub-species (I, II, IIIa, IIIb, IV, VI).
• S. bongori – formerly subspecies V (20 serotypes).
6 subspecies of S. enterica.
• >2500 serotypes of S. enterica subsp. Enterica.

11. • Oral or intravenous rehydration.
• Generally antibiotics are not recommended.
– Agents do not shorten course of illness.
– Antibiotic resistance increasing problem.
– Antibiotics can prolong excretion of Salmonella.
– Exceptions include high risk groups.
• Underlying disease or complicated medical history
• Young children
• Disseminated infection
• Antibiotics
– Objective is to treat or prevent recurrence of bacteremic disease .
– Fluoroquinolones
– Azithromycin
– TMP-SMX
Classification - Salmonella species
and serotypes

12. Ian McHardy, Ph.D.
Ian McHardy, Ph.D. is second year CPEP
fellow at UCLA. Dr. McHardy studied
aspects of the human microbiome for his
Ph.D. thesis and postdoctoral fellowship.
His research interests involve linking
bioinformatics with basic and clinical
research to drive improved diagnostics,
disease monitoring, and therapies for
infectious diseases.
Photo Credit: Ian McHardy, Ph.D.