Salmonella enterica

serovar Typhi
Prof. Mochammad Hatta, MD, Ph.D, Clin Micro (Cons) Dept Microbiology, Molecular Biology and Immunology Laboratory, Fac.Medicine, Hasanuddin University, Makasssar, Indonesia

INTRODUCTION
Discovered in 1880 & named after Daniel Salmon, the pathologist who first isolated the organism from porcine intestine.

Salmonella is a motile, gram-negative, rodshaped bacteria, which is a leading cause of bacterial food-borne diseases. Of the 2000 strains recognized, human infection are caused mainly by 5 serotypes, typhi, paratyphi, typhimurium, choleraesuis & enteritidis.

SALMONELLOSIS
Salmonella typically produces 3 distinct syndromes: food poisoning, typhoid fever & asymptomatic carrier state.

Salmonella gastroenteritis manifest as vomiting & diarrhea within 6-48 hours after ingestion of food or drink contaminated with bacteria.
It is self-limiting, treatment is by water & salts replacement. Antibiotics are not usually needed.

TYPHOID FEVER
Typhoid fever is the most serious salmonella infection with significant morbidity & mortality. Caused by salmonella typhi & paratyphi. Incubation period is 1-2 weeks. Salmonella has somatic (O antigen) & flagellar H antigen. The O antigen is more specific for serologic testing.

FREQUENCY
An estimated 15-30 million cases of typhoid fever occur globally each year. The disease is endemic in many developing countries in Asia, Central America & Africa. Outbreak of typhoid fever have been reported recently from Eastern Europe. Incidence in Sudan is not exactly known, but estimated as 50 per 100,000 people/year.

PRECIPITATING FACTORS
Defects in cellular-mediated immunity (AIDS, Transplant patients & malignancy). Defects in phagocytic function (malaria, histoplasmosis & schistosomiasis). Splenectomy or functional asplenia (sickle cell dis) Low stomach PH ( patients on anti-ulcer drug). Prolonged use of antibiotics (altered gut flora). Injured gut barrier (bowel disease or surgery).

MORTALITY & MORBIDITY

Infection with nontyphoidal salmonella produces self-limiting gastroenteritis and food poisoning. Whereas mortality caused by typhoid fever is rare in western countries, it is associated with significant mortality & morbidity in tropical countries (10-30%).

Dehydration is the most common complication of typhoid fever, but serious intestinal & extraintestinal complications may occur.

 Disease : Demam Tifoid in Indonesian (Typhoid Fever)

Gastrointestinal infection -- endemic in Indonesia--developing countries -- environmental/sanitation/hygiene relationship

(Hattta M, et al. International Journal Tropical Medicine, 2009)

Family : Enterobacteriaceae -- Gram negative -- non-sporing bacillus -- 2-4 -- motile -- long peritrichous flagella -- aerobe/ facultative anaerobe -- temperature 15o- 41o & optimum 37o C) Colonies on MacConkeys medium & SS agar

Biochemical reactions: Glucose & mannitol: fermentation (+) &

gas (+) -- lactose & sucrose : fermentation (-)
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PATHOPHYSIOLOGY
After ingestion salmonella must survive the stomach acidic PH & colonize small intestine. Salmonella then attach to & penetrate the gut mucosa resulting in diarrhea from direct mucosal damage & by action of exotoxins. Another portal of entry is invasion of lymphoid tissue in the GIT (peyer patches) & multiplication within macrophages leading to bacteremia.

 Viability : death point : 56o C In soil survival for 6 weeks Pathogenesis : infection by ingestion --- small intestinal via lymphatics --- mesenteric glands -- multiplication --- blood via thoracic duct --- bacteriaemic phase ( 1 - 10 days) : infection

liver, gall bladder, spleen, kidney & bone marrow.

Gall bladder --- invasion lymphoid tissue -- Peyers patches & lymphoid follicles -- acute inflammatory reactions --- ulcer

haemorrhage -- perforation & necrosis

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POSITIVE CULTURE AND IgM ANTIBODY RESPONS IN TYPHOID FEVER

100 90 80 70 60 50 40 30 20 10 0 1 2 3 4 5 6 7 8 Weeks

Blood Faeces Urine IgM

% Positive

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DIFFERENTIAL DIAGNOSES
Cryptosporidiosis Campylobacter infection Cyclospora Listeria monocytogenes Escherichia Coli infection Shigellosis

LAB FINDINGS
Salmonella can be grown from blood or bone marrow in the 1st week, from stool in the 2nd week & from urine in the 3rd week. Special media are needed for transport & for culture.
leukopenia is typical but WBC may be normal. Widal test is not diagnostic, titer > 1:320 or 4 fold increase in titer support the diagnosis.

Laboratory diagnosis

Typhoid fever

Polymerase Chain Reaction (PCR) Culture of blood or bone marrow 80% during first week Culture of urine or stool in presence of characteristic clinical picture Serology test antibody test against somatic (O) or flagellar (H) antigen

Deteksi Salmonella typhi dengan Nested PCR

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Typhoid fever (nested) ST1 : 5-ACT GCT AAA ACC ACT ACT-3 ST2 : 5-TTA ACG CAG TAA AGA GAG-3 ST3 : 5-AGA TGG TAE TGG CGT TGC TC-3 ST4 : 5-TGG AGA CTT CGG TCG CGT AG-3 (M. Hatta & Henk L Smits. American J. Tropical Medicine & Hygeine, 2007)

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Hasil nested PCR S.typhi dari penderita demam tifoid

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MDR PCR product S.typhi Vietnam and Indonesian isolated Vietnam Indonesia

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MDR PCR product S.typhi Vietnam isolated

941 bp 819 639

310

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PCR for the detection of S. typhi specific DNA in blood, stool and urine samples from patients with suspected typhoid fever .

Patient group

No (%) of patients with the following result Blood Faeces Urine Neg Pos Neg Pos Neg

Pos

Culture positive Culture negative

1 (1) 71 (99) 21 (45) 26 (55)

16 (67) 7 (41) 2 (100)

8 (33) 10 (59) 0 (0)

22 (38) 11 (28) 10 (100)

36 (62) 28 (72) 0 (0)

Non-typhoid patients 12 (100) 0 (0)

(INCO-DC EC Research project Report, 2002)

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CONSTRAINTS OF PCR TECHNIQUE

1. Quite expensive 2. Need special equipment 3. Need high skill and laboratory 4. Sophisticated

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Salmonella bacteria on MacConkey agar Lactose-positive bacteria show pink colonies (upper left) Lactosenegative bacteria have colorless colonies (lower right)

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Biochemical reactions for identification of S. typhi by the API 20E procedure

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growth on bismuth sulfite agar

Salmonella typhi after

Black colonies of

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Dipstick for Typhoid Fever

Procedure Add 5l serum to 250l detection reagent Incubate dipstick for 3 hours Rinse with tap water Read by visual inspection
Result

(Mochammad Hatta, et al. American J. Tropical Medicine & Hygiene, 2002)

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Mochammad Hatta

Dipstick for Typhoid Fever

Procedure Add 5l serum to 250l detection reagent Incubate dipstick for 3 hours Rinse with tap water Read by visual inspection Result
Patients with clinical suspicion of typhoid fever from Makassar, Indonesia

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Control Test

CTD, Ho Chi Minh City, Viet Nam Comparison of tests

Test IgM ELISA 1:400 IgG ELISA 1:1.600 IgA ELISA 1:200 Widal O 1:400 Widal H 1:200 Dipstick Sensitivity (%) 75 68 52 47 60 77
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Typhoid Fever Dipstick

Specificity (%) 94 92 95 93 98 95

Typhoid Fever Dipstick

Semarang, Indonesia
Patient group, culture result Dr. Kariadi Hospital
(bone marrow culture)

Number positive (%) / total

S. typhi positive S. typhi negative

38 (70.4) / 54 0 (0) / 2

3 district hospitals (blood culture) S. typhi positive S. typhi negative

32 (86.5) / 37 2 (7.7) / 26

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Typhoid Fever Dipstick Makassar, Indonesia

Patient group Suspects Clinical diagnosis: typhoid S. typhi culture positive S. paratyphi culture positive Culture negative Clinical diagnosis: other Culture negative Hospital controls School children
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No. positive (%) / total 85 (47.5) / 179 73 (65.2) / 112 4 (66.6) / 6 8 (13.1) / 61 0 (0) / 64 0 (0) / 259 2 (1) / 194

Typhoid Fever Dipstick Makassar, Indonesia

Follow-up
Sample
DPO

No. positive (%) / Total 30 (76.9) / 39 32 (82.1) / 39 38 (97.4) / 39 2 (4.3) / 47 36 (76.6) / 47 39 (83.0) / 47

S. typhi culture positive

First Second Third

8 15 29 6 13 27

S.typhi culture negative

First Second Third

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Typhoid fever Culture and Dipstick

Assay Culture Dipstick Sensitivity Specificity 65.9% 47.5% 100% 95% PPV 100% 92% NPV 74% 65%

Dipstick: finger prick blood, same day result

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Rapid test for typhoid fever

80% sensitivity compared with blood culture PPV (92%) and NPV (64%) somewhat lower than that of culture Same day result Easy to perform High stability of components
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DIPSTICK FOR DETECT IGM ANTIBODIES

1. Simple and rapid 2. Required no equipment 3. Highly stable reagents 4. Low cost 5. Easy to applied in field

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TYPHOID Lateral Flow

Principle

Immunochromatographic strip assay

Test Control

Sample pad / blood cell separation filter

Conjugate pad

Detection strip

Sink

TYPHOID Lateral Flow

Method

Add 5l serum

Add 130l sample fluid

Wait 10 minutes Read result

Control line Test line

Sample well

Typhoid Fever Latex Agglutination

5 seconds

15 seconds

45 seconds

> 60 seconds
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What is S.typhi ?

A bacteria Causes typhoid fever that affects 16 million people annually and causes 600,000 fatalities Has evolved the ability to spread from the intestine to the deeper tissues of humans, including the liver, spleen, and bone marrow

What is it? Cont

Resistant to many drugs Closely related to Salmonella typhimurium (also already sequenced), classified under the same species as Salmonella typhi Difference is that S. typhi causes typhoid fever and can only infect humans, whereas S. typhimurium causes food poisoning and can affect almost all animals

Where and When?

Sequenced by the Sanger Institute

On November 7, 2001
Mochammad Hatta. How diversity flagella variants of S. Typhi strains in Indonesia Archipelago?. Wellcome Trust Advanced Course: Molecular

Basis of Bacterial Infection: Basic and Applied Research Approaches. Wellcome Trust Genome Campus, The Sanger Institute,

Hinxton, Cambridge, United Kingdom (UK), 11-17 May 2008. page 12-13.

Why?

It affects millions of people and sequencing the genome can help us find a way to block its transmission in humans, eradicating it altogether Can help improve diagnostic tools and vaccines

Why?

Multiple drug resistance (MDR) is a emerging problem in treating infectious diseases Salmonella typhi is one example of MDR microorganism It is resistance to fluoroquinolones, the most effective antimicrobials for the treatment of typhoid fever

So..?

Since salmonella typhi is an example of an emerging MDR microorganism, studying this genome can contribute to the understanding of how such microorganisms adapt rapidly to new environmental changes that are presented by modern human society.

Some Statistics

Chromosome sequence is 4,809,037 bp in length C+G content of 52.09% 4,599 protein-coding genes (402 of these are pseudogenes)

Side Note:
Pseudogene: once functional stretches of DNA that have been inactivated by mutation

Some Results

The genome shows hundreds of deletions and insertions, resulting in MDR Found the plasmid in Salmonella typhi that encodes resistances to all of the first-line drugs used for the treatment of typhoid fever Many other genes responsible for resistance in drugs were indentified

Side Note

Plasmid: A piece of symbiotic DNA, mostly in bacteria but also in yeast, not forming part of the normal chromosome DNA of the cell and capable of replicating independently of it. Plasmids carry a signal situated at their replication origin dictating how many copies are to be made, and this number can be artificially increased.

Results Cont

Salmonella typhis genome gives us hints as to why it only infects humans Because it has 204 pseudogenes. Working versions of these genes were discarded during typhis evolution for its current habitat in humans Vs. typhimurium only has about 40 pseudogenes

Results Cont

Both typhi and typhimurium have hundreds of genes that are different. This is very surprising because these two organisms are classified as a single species

CLINICAL PICTURE
Symptoms begin with sudden onset of highgrade fever, headache & dry cough. Fever is swinging or may show step ladder pattern & patient initially feel well & mobile. Abdominal pain & toxicity follow soon & by the end of 1st week spleen is palpable & pink, discrete, skin rash appears over the trunk. Constipation is more common than diarrhea which is usually greenish in color (pea soup).

CLINICAL PICTURE/2
Abdominal tenderness & hepatomegaly occur in 50% of patients.
The pulse is relatively slow in relation to fever (Paget sign). The tongue is coated with free margins & halitosis may be present. The sweat of some patients smell like yeast.

CLINICAL PICTURE/3
The 3rd week of illness is the usual time for complications in the untreated patients. Local gut as well as systemic complications may occur. Serious infections may progress rapidly to drowsiness & coma which is usually fatal (coma vigil). Mortality is unlikely after the 4th week & patients may become carrier if not treated.

LOCAL COMPLICATIONS
Intestinal hemorrhage
Intestinal perforation Paralytic ileus

Zenker degeneration of abdominal muscles

SYSTEMIC COMPLICATIONS
Endocarditis Arteritis & arterial emboli Cholecystitis

Hepatic & splenic abscesses

Pneumonia or empyema

Osteomyelitis & septic arthritis

Meningitis Urinary tract infection

TREATMENT
Medical care include rehydration, antipyretics & antibiotics. Drugs of choice are Ceftriaxone & ciprofloxacin but Cotrimoxazole & Chloramphenicol are still used in developing countries. Ampicillin kills bacilli hiding in the bile & hence prevents or reduce the carrier state. Chronic resistant carrier state may necessitate cholecystectomy. Surgical care may also be needed in patients with intestinal complications.

NURSING CARE
Isolation & barrier nursing is indicated Notification of the case to the infection control nurse in the hospital. Trace source of infection.

continue breastfeeding infants & young children and give ORS & light diet for other patients in the first 48 hours.

PREVENTION
Education on hygiene practices like hand washing after toilet use & avoidance of eating in non hygienic restaurants.

Proper handling & refrigeration of food even after cooking.

Salmonella TAB vaccine is available but affectivity is low (50% claimed protection). Antibiotic prophylaxis is not needed for house-hold contacts.

PROGNOSIS
With early diagnosis and prompt treatment most patients with typhoid fever will recover in due time. Fever & toxicity subsides within 72 hours of antibiotic treatment. Mortality is > 50% in untreated severe typhoid fever particularly in children & elderly. Recrudescence is rare but chronic carrier state is reported in 10% of patients.

TRANSMISSION
Infection follows ingestion of contaminated food or water. Meat, poultry, eggs & diary products are frequent sources.

Pets, domestic animals and infected human are potential reservoirs. Person to person & animal to human transmission is recognized.
In healthy humans a dose of about one million bacteria is necessary to produce symptoms.

References

Mochammad Hatta, Mirjam Baker, Stella van Beer, Theresia H Abdoel, Henk L Smits. Risk factors for clnical typhoid fever in villages in Rural South Sulawesi, Indonesia. International Journal of Tropical Medicine. Vol 4 (3): 91-99, (2009) Mochammad Hatta and Ratnawati. Enteric fever in endemic areas of Indonesia: an increasing problem of resistance. J. Infection Developing Countries (JIDC). Vol 2(4); 298-301 (2008) Rob Pastoor, Mochammad Hatta, Theresia H. Abdoel, Henk L. Smits. Simple, rapid and affordable point-of-care test for the serodiagnosis of typhoid fever. J. Diagnostic Microbiology and Infectious Disease. Vol 61:(2);129-134, Feb (2008). Mochammad Hatta and Henk L Smits. Detection of Salmonella typhi by nested Polymerase Chain Reaction in blood, urine and stool samples. American J. Tropical Medicine Hygiene.vol : 76;139-143 (2007). Theresia H. Abdoel, Rob Pastoor, Henk L. Smits, Mochammad Hatta, Laboratory evaluation of a simple and rapid latex agglutination assay for the serodiagnosis of typhoid fever. Transactions of the Royal Society of Tropical Medicine and Hygiene. vol. 101 (10); 1032-1038 (2007) Mochammad Hatta, Marga D.A Goris, Evy Heerkens, George C Gussenhoven, Jairo Goosken, Henk L Smits. Simple dipstick assay for the detection of Salmonellla typhi-specific immunoglobulin M antibodies and the evolution of the immune response in patients with typhoid fever American J. Tropical Medicine and Hygiene. vol 66: no 4; 416-421 (2002). Mochammad Hatta, Mubin Halim, Theresia Abdoel, Henk L. Smits. Antibody response in typhoid fever in endemic Indonesia and relevance of serology and culture to diagnosis. Southeast Asian Journal of Tropical Medicine and Public Health. vol 33: no 4; 182-191 (2002).