Emerging Bacterial Diseases-Melioidosis

1. Melioidosis
Presented by: Babita Sharma
Roll number: 02
3rd semester Medical microbiology

2. Introduction
 Melioidosis is an infectious disease caused by bacterium Burkholderia
pseudomallei( previously known as Pseudomonas pseudomallei) that can infect
humans or animals.
 Also called Whitmore’s disease, pseudoglanders, night cliff disease, paddy-field
disease.
 It has several forms like, the formation of skin abscess, sepsis, septic shock,
abscess formation in several internal organ and pulmonary disease.
 Human acquired infection by inhalation of infected aerosol, inoculation of
contaminated material or excreta of infected animals and direct contact with soil.
 Melioidosis is primarily a disease of rats, but also occurs in guinea pig, rabbits,
goats and dog.
 Melioidosis is clinically and pathologically similar to glanders disease.

3. History
 This disease, now termed melioidosis, was named from Greek word “ Melis ”(
distemper of asses) and “eidos”(resemblance) by Stanton and Fletcher in 1932.
 Melioidosis was first discovered in Burma( now Myanmar) by Whitemore and
Krishmaswami in 1911.
 Also reported in Malaysia and Singapore in 1913 and then Vietnam in 1925.
 First diagnosed case in nepal was in 2005.
 Potential use of B. pseudomallei as a biological Weapon.
 Glanders together with Anthrax was implicated in the first modern era as
biological warfare (1915-1918) to infect horses in the united state, Romania,
Spain, Norway and Argentina.

4. Reason to use in Bioterrorism
 High mortality rate
 Easy transmitability
 Short incubation period
 Difficulty is diagnosis and treatment
 Ability to survive outside its natural environment
 Intrinsic antibiotic resistance

5. Epidemiology
 Melioidosis is predominantly disease of tropical climates. occurs worldwide but
most common in Southeast Asia ,Australia , Thailand Africa, India and china.
 A modelling study estimate that there are 165,000 cases of melioidosis in human
per year worldwide, of which 89,000 (54%) are estimated to be fatal.
 Melioidosis is prevalent in the northern Australia and northeast Thailand, where
the annual incidence is up to 50 cases per100,000 individual.
 Bacteria occurs as an environmental saprophyte found in the soil, rice paddies
and muddy water particularly common in moist clay soil.
 Polluted and contaminated atmosphere contribute to spread.

6. Bacteriology
Morphology
 Burkholderia pseudomallei is a Gram -negative, Motile, obligate aerobic, non
spore forming bacillus that is straight or slightly curved.
 Measure about 2-5µm in length and 0.3-0.8 µm in diameter.
 Typical bipolar safety pin like appearance in the Methylene blue stain.
 Facultative intracellular pathogen.

7. Agent and Reservoir
 Agent: Burkholderia pseudomallei
 Reservoir:
 Soil, rice paddies and muddy water.
 Unchlorinated water supply and drinking water in rural area.
 B. pseudomallei can survive in extreme conditions, such as in distilled (without
nutrients) water (for ≥16 years), 40% soil moisture content for longer than 2
years and 0% moisture content around 30 days survive.
 No animal reservoir.

8. Mode of transmission
 Inhalation
inhalation of Dust, contaminated soil and aerosol containing bacteria.
 Ingestion
by consuming contaminated water, Unchlorinated domestic water , ingestion
of food and water contaminated with infected animal excreta and soil.
 Inoculation
inoculation during work on a soil through injuries and puncture of skin of
farmer.
 Breast milk
Transmitted to infants through breast milk from infected mothers.
 Direct contact with contaminated soil and water.
 Vector born transmission via mosquito (Aedes aegypti) and rat flea (Xenopsylla
cheopsis)
 Vertical, sexual, Human to human and animal to human transmission is rare.

9. Risk factor
Diabetes mellitus
Alcohol use
Chronic lung disease
Chronic renal disease
Immunocompromised patients
Cancer patients

10. Virulence Factor and pathogenesis
Virulence factor function
Flagella host cell attachment
Pilli host cell attachment
Cps Antiphagocytic, Biofilm production
T3ss Intracellular invasion and escape phagocytosis
T6ss1 Intracellular invasion and intracellular spread
BipB and BipD Intracellular invasion
BipC Adhesion, invasion, actin formation
INOS Intracellular survival
BIMA Actin polymerization

11. Pathogenesis
 Bacteria first enter at a break in the skin or mucous membrane and replicate in
epithelial cell.
 Use flagellar motility to spread and infect various cell types.
 T3ss transport the protein across the cell membrane which causes invasion.
 Attachment via adhesion protein, including the type IV pilus protein pilA and
adhesion protein BoaA and BoaB.
 Enters host cell by endocytosis.
 Bacteria replicates in both phagocyte and non-phagocyte cell intracellularly
which causes the lysis and infect the adjacent cell.
 Bacteria trigger the autophagy by the activation of NOD2 ie intracellular
pathogen recognition receptor protein that causes bacteria killing.

12.  T3SS inject effector proteins( BopA )which help to escape and protection by
host autophagy. macrophage lysis is mechanism for bacterial survival.
 Replication occur in host cytoplasm.
 Inside the host cell, polymerization of host actin propel bacteria to move
forward until reaches to CNS.
 Then, profusion occur which fuses the neighbouring cells forming
multinucleated giant cell(MNGC) that results in plague and the damage of host
cell.
 Plaque (a central clear area with a ring of fused cells) that provide shelter for
bacteria for further replication or latent infection.
 Direct cell to cell spread occurs in blood stream causing sepsis and infection in
antigen presenting cell that transport bacteria to the lymphatic
system(secondary spread).

13.  Bacteria can remain dormant for 19-29 years until imunosupression or other
host stress reactivates bacteria proliferation.
 Toxin anti-toxin system host immune response and selective pressure of
antibiotics are the factors contributing to latency.

14. Clinical manifestation
The disease shows different stage such as acute, local and chronic infections. The
incubation period is variable. It ranges from 2 day to as high as to many years.
Acute melioidosis
 Development of nodule, pain or swelling ,ulceration, abscess at the site of
infection of bacteria in the skin.
 Bacteria subsequently spread, causing secondary lymphangitis, reginal
lymphangitis, fever and myalgia.
 Progress rapidly to acute septicaemia with high mortality rate.
 Acute blood stream infection is most commonly seen in patients with HIV,
diabetes, renal failure etc. the condition results in septic shock.

15. Pulmonary infection
 Manifests as mild bronchitis to severe pneumonia.
 The condition is associated with high fever, headache, chest pain, anorexia and
general myalgia.
 Productive and non productive cough with normal sputum is typical
manifestation.
Chronic suppurative infection
 It is associated with multiple caseous or suppurative foci of infection in several
organs including joints, skin, lymph nodes, spleen, lungs, liver and brain.
 Bacteria remains as intracellular pathogens of the reticuloendothelial system,
which contributes to long latency and reactivation of the infection. Hence this
disease is called as Vietnam time bomb disease.

17. Clinical sign in animal
 Widely vary within a species, depending on the site of infection ; range from
acute to chronic.
 Subclinical infection is common.
 Single or multiple suppurative or caseous nodules/abscesses formation.
 Mastitis in goats
 The respiratory infection in sheep.
 Fatality occur when vital organs are infected.

18. Laboratory diagnosis
 Sample specimen
 Sputum
 BAL
 Blood or bone marrow
 Urine
 Throat swab
 Pus and wound swab
 Skin lesions
 Rectal swab

19.  Microscopy :
Gram stain : gram negative
Methylene blue stain :bipolar safety pin appearance
 Culture(gold standard)
 Not fastidious and grow on a large variety of culture media( BA,CA,MA etc).
 Ability to grow on 42°c. culture typically positive in 24 to 48 hours.
 Ashdown’s medium, Brukholderia pseudomallei selective media, francis media
and modified Ashdown’s medium are used for selective isolation.
 Ashdown’s medium contains crystal violet and gentamicin as selective agent. It
usually produce irregular-edge, rough, flat wrinkled purple colonies.
 Required BSL 3 laboratory for handling of organism.

20.  Resistance pattern
 Gentamicin and colistin resistance
 Amoxicillin-clavulanate sensitivity
 Biochemical reaction
 motility : positive
 Oxidase : positive
 Nitrate reduction: positive
 Indole : negative
 Methyl Red : negative
 Voges-Proskauer : Negative
 H2S : negative
 TSI : A/K
 Lactose and maltose fermenter

21.  Serological test
Rapid antigen detection
 Latex agglutination : Target LPS( sensitivity-94% and specificity—83%)
:Target exopolysaccharide (sensitivity-98.7% and specificity-97.2%)
 Immunofluorescence assay(IFA): Target exopolysaccharide
blood: sensitivity 100% and specificity 99.6%
Nonblood sample: Sensitivities 32.7% (respiratory sample ) to 50%(pus)
 Lateral flow assay: Target capsular polysaccharide
Sensitivity-98.7% and a specificity-97.2%
 Indirect hemagglutination assay(IHA): titer≥ 1:160 in the absence of a positive
culture is therefore regarded as supportive rather than definitive.
Poorly defined antigens from strain of B. pseudomellei absorbed to red blood cell.
 ELISA: commercial kit for melioidosis appears to perform well.

22.  Molecular technique
 PCR: Detection of pathogenic B. pseudomallei DNA from a clinical specimen.
 DNA Sequencing
Radiological test
 CXR: for diagnosis of pulmonary melioidosis.
 CTscan : used to diagnosis abscesses in the liver and spleen.

24. Treatment
Antimicrobial therapy is separated into the initial intensive phase and the subsequent
eradication phase. No validated vaccine are available till now.
Intensive phase
 Ceftazime, 2g( child, 50mg/kg upto 2gm), every 6 hours.
 Meropenem, 1g( child, 25mg/kg upto 1 gm), every 8 hours.
Eradication phase
 Trimethoprime- sulfamethoxazole ( child 6/30mg/kg upto 240/1,200mg orally,
every 12 hours.
 Folic acid, 5 mg (child, 0.1mg/kg upto 5 mg) orally daily.

25. Prevention and control
 Wearing personal protective equipment such as gloves and suitable clothing for
high risk group like agricultural worker.
 Disinfection (chlorination and chloramination) of the drinking water supply.
 Mechanization of agricultural activities in disease endemic area.
 Awareness raiser among veterinary and human health authorities.
 Avoid contact with animal urine, infected animals or an infected environment
e.g. waterlogged places where infected animals may have urinated.

26.  Drink only safe or boiled water during the rainy season, especially in flood-
prone areas.
 Practice good personal hygiene, washing hands before eating and after
defecating.
 Consult a physician for prophylactic use of antibiotics during flooding times.
 Protect the water supply from animal contamination.
 Use of disinfectant such as 70% ethanol, benzalkonium chloride, iodine etc.

27. Melioidosis in Nepal
 Shrestha N.K et al (2005) reported first case of melioidosis in a patient of 34 years
old male who had returned to Nepal from Malaysia.
 According to Shrestha N et al(2019) there was 2 cases reported at Tribhuvan
University Teaching Hospital. Both of them were diabetic male patients.
 According to Chaudhary R et al (2019) reported the case of cerebral melioidosis in
22years old male at Tertiary Care Hospitals . Who was also an soldier from
commando Training Academy.

28. Bibliography
 Tille, Patricia M. (2014). Bailey and Scott’s Diagnostic Microbiology. 14th edition. St.
Louis, Missouri: Elsevier.
 Parija SC (2012).Textbook of Microbiology and Immunology. 2nd edition. Noida, India:
Elsevier.
 WHO (2014). A Brief Guide to Emerging Infectious Diseases and Zoonoses.
 Shrestha NK, Sharma SK, Khanal B, Dhakal SS (2005). Melioidosis imported into
Nepal. Scandinavian Journal of infectious disease 37(1):64-80.
 Mukhopadhyay C, Shaw T, Varghese GM, Dance DAB(2018). Melioidosis in South Asia
(India, Nepal, Pakistan, Bhutan and Afghanistan). Tropical Medicine and Infectious
Disease.3(2):51
 Wiersinga WJ, Virk HS, Torres AG et al (2018). Melioidosis. Nat Rev Dis Primers ;
4:17107.
 https://www.uptodate.com/contents/melioidosis-epidemiology-clinical-manifestations-
and-diagnosis.
 https://wwwnc.cdc.gov/eid/syn/en/article/21/2/14-1045.

29.  Shrestha P, Adhikari M, Pant V, Baral S, Shrestha A, Bashyal B and Sherchand
JB(2019). Melioidosis misdiagnosed in Nepal. BMC infectious disease,19:176.
 Chaudhary R, Singh A, Pradhan M, Karki R, and Bhandari PB(2019). A fatal case
of cerebral Melioidosis. MJSB, 18:64.

30. Thank you