Burkholderia pseudomallei is the causative agent of melioidosis, a serious disease of man and animals that occurs primarily in S.E. Asia, N. Australia and other ...
Bacteraemia due to Burkholderia pseudomallei in Nakhon Phanom, Thailand Ornuma Sangwichian1, Tanitta Channa2, Payaowadee Abthaisong2, Thummaporn Wongpadung2, Chutathip Techawetcharoen2, Sompong Photin2, Sujitra Nimnuan2, Pornpan Bungkajana2, Sumalee Boonmar3, Toni Whistler3 1 International Emerging Infection Program , Nakhon Phanom, Thailand; 2Project staff of International Emerging Infection Program, Nakhon Phanom, Thailand; 3 International Emerging Infection Program, Global Disease Detection, Nonthaburi, Thailand
Introduction
Materials and Methods
Burkholderia pseudomallei is the causative agent of melioidosis, a serious disease of man and animals that occurs primarily in S.E. Asia, N. Australia and other tropical areas. B. pseudomallei is an environmental Gramnegative saprophyte, present in wet soil and rice paddies in endemic areas. The highest documented infection rate is in northeastern Thailand, where melioidosis accounts for 20% of all community-acquired septicemias [1]. Disease occurs after bacterial contamination through breaks in the skin, or by inhalation after contact with water or soil. It is also the most common cause of fatal community-acquired pneumonia [2].
Transport to IEIP Nakhon Phanom lab at appropriate temperature (15-30°C)
- Pediatric Aerobic (PF) for child pa ents age < 5 years - 20 mL peptone enriched TSB supplemented with BHI solids and ac vated Charcoal - Specimen type : Blood - Specimen volume : 4 mL -MB blood system (MB) for suspected Mycobacterium infec on -29 mL supplemented Middlebrook 7H9 broth with glycerol and SPS. - Enrichment Fluid ( Bovine serum albumin, sodium chloride, oleic acid and saponin in water) - Specimen type :Whole blood - Specimen volume : 5 mL
Incubate in BacT/ALERT 3D machine (Biomerieux,USA) at 36°C - 5 days for FA and PF bo le - 42 days for MB bo le
The aim of our study was to investigate the prevalence of Burkholderia pseudomallei bacteremia in hospitalized patients in Nakhon Phanom and examine the antimicrobial susceptibility over a one year period (2012).
Results
Table 2. List of pathogens isolated from hemoculture in NP over a single year, 2012 Pathogen Escherichia coli Streptococcus species Burkholderia pseudomallei Klebsiella pneumoniae Staphylococcus aureus Gram-nega ve bacillus Acinetobacter baumannii Salmonella species Enterococcus species Enterobacter species Klebsiella species Streptococcus viridans group Streptococcus pneumoniae Vibrio species Other pathogens* Total Isolates
Isolates 303 139 127 101 74 72 27 26 25 18 17 17 13 12 785 1756
% 17.3 7.9 7.2 5.8 4.2 4.1 1.5 1.5 1.4 1.0 1.0 1.0 0.7 0.7 44.7 100
*Other pathogens included mold, fungi, Serratia species, Pseudomonas aeruginosa, Aeromonas and Proteus species
The strip has an exponential scale on it for determining MIC, it is impregnated with the antibiotic of interest, in this case Ceftazidime.
- 30 mL peptone enriched TSB supplemented with BHI solids and ac vated Charcoal - Specimen type : Blood or SBF - Specimen volume : 10 mL
Objective
Figure 2. B. pseudomallei colony morphology after sub-culture on MacConkey agar (left) and on Chocolate agar (right) after incubation at 35°C for 48 hrs . The B. pseudomallei colonies appear dry and wrinkled and have a metallic appearance. They also possess an earthy odor.
Figure 3. Gram stain of B. pseudomallei showing Gram negative rods with a characteristic “Safety pin” appearance (also known as bipolar staining (inset).
Figure 1. Photograph of Etest results showing the bacterium is susceptible to Ceftazidime. (Arrow showing MIC = 1.0 µg/mL).
Gram stain ,sub culture on Media plates (Sheep Blood Agar, Chocolate Agar and MacConkey Agar)
BacT/ALERT media bo le - FAN Aerobic (FA) for adult pa ents
Appropriate antimicrobial agents should be administered immediately on suspicion of a meloidosis diagnosis, since delays or ineffective therapy is associated with a very high mortality rate with septicemia.
• From January-December 2012, blood was collected from a total 9,617 pneumonia patients from 11 hospitals in Nakhon Phanom province. Of these 52% were men, and 53% were over the age of 50 years. • All blood specimens were screened for the presence of bacterial pathogens using an automated blood culture, the BacT/ALERT system (Biomerieux, USA). B pseudomallei was isolated from 127/9617 specimens (Table 2). • Morphology of bacterial colonies on sub-culture on different types of agar plates (shown in Fig. 2) is used as an indicator that B. pseudomallei is the pathogen isolated, this is conrmed by Gram stain (Fig. 3) andbiochemical testing (Fig. 4). • Fig. 1 illustrates the result of an E test for Ceftazidime susceptibility of a B. pseudomallei isolate. The rate of antimicrobial susceptibility to Cotrimoxazole was 93% and Ceftazidime 100%.
Alarm posi ve bo les
9,617 blood bo les (FA,PF and MB) collected at 11 hospitals in Nakhon Phanom
Inden fied by biochemical tests (Triple Sugar Iron (TSI), Mo lity, Indole, Lysine decarboxylase/deaminase (LDC/LDA), Citrate, Urea and Oxida on fermenta on (OF) - glucose) An microbial suscep bility test using the Epsilometer -test (or E-test, an agar diffusion method) using Mueller-Hinton agar plates (Biomedia): • A lawn of bacteria is spread and grown on an agar plate, and the E-test strip (a rectangular strip impregnated with either Ce azidime or Cotrimoxazole ) is laid on top the agar. • The an bio c diffuses out into the agar, producing an exponen al gradient of the drug to be tested. A er 24 hours of incuba on, an ellip cal zone of inhibi on is produced and the point at which the ellipse meets the strip gives a reading for the minimum inhibitory concentra on (MIC) of the drug (Fig . 1 and Table 1)
• The number of isolates made of B. pseudomallei compared to the total number of blood cultures performed at each of the 11 hospitals is shown in Fig. 5. Nakhon Phanom, the large provincial hospital, had the largest number of blood cultures, with a B. pseudomallei isolation rate of 1.1%. The highest prevalence was seen at Renoonakorn hospital (RN, Fig. 5) where 2.4% of all blood cultures were positive for B. pseudomallei. • Exploring the time to positive alarm, hemoculture bottles (in which B. pseudomallei was identied) alarmed 1224 hrs after inoculation in 48% of the bottles (Fig. 6), while 40% were “alarm positive” at 24-48 hours. • The infection rate is highest in those >50 years old (Fig. 7), and more men are infected than women (57%) (Fig. 8).
Table 1: MIC for E-test interpretation for B. pseudomallei as explained in the Clinical and Laboratory Standards Institute (CLSI) guidelines (M45-A2,2012). An microbial Agent
MIC Interpreta on Standard An microbial (µg/mL) Sensi ve Intermediate Agent Sensi ve Intermediate Resistant (S)(S) (I) (I) ®
Ce Ce azidime
TSI
Mo le
Indole
LDC
LDA
Citrate
Co-Trimoxazole
Triple Sugar Iron
Oxida on
Fermenta on
Tube
1
2
2
3
3
4
5
6
6
Results
A/N
+
-
+
-
+
-
+
-
Figure 6. Time to alarm positive in BacT/ ALERT bottles for B. pseudomallei isolates. A total of 127 alarm positive bottles were identied.
Blue
Pink
Yellow at top of tube
Yellow at bo om
Biochemical test result interpreta on
Pos
Slant Acid (A )= yellow No Change (N) TSI bu Acid (A)= yellow
Neg
Migrates
Alkaline (K) = Pink from stab line
Pink/Red on surface
No change
Red/Purple
No migra on
Alkaline (K )= Pink from stab line
No change
Yellow
No change
No change
No change
No change
No change
No Change (N)
Figure 4. Biochemical tests results for B. pseudomallei with a table (below) explaining the result interpretation.
Figure 5. Number of B.pseudomallei positive blood cultures by hospital during 2012
= 8
< =82 32 >4
Conclusion
OF-Glucose
Urea
azidime
Co-Trimoxazole
Figure 7. Break down of patients by age from which B. pseudomallei was isolated. Test
MIC Interpreta on Standard (µg/mL)
Figure 8. Sex of patients from which B. pseudomallei was isolated.
• Burkholderia pseudomallei is an important pathogen in NE Thailand. It was the third most common pathogen identied by hemoculture from pneumonia patients in the Nakhon Phanom province in 2012. • Almost all patients were agricultural workers who are at risk through contact with contaminated soil and water, especially through skin abrasions. It is important for public health personnel to share their knowledge of this disease and how to prevent infection with this high risk population. • Antimicrobial susceptibility testing showed very little resistance to Cotrimoxazole and none to Ceftazidime.
References 1. Chaowagul W, White NJ, Dance DA, et al. Melioidosis: a major cause of community-acquired septicemia in northeastern Thailand. J Infect Dis 1989; 159(5):890-9. 2. Duplessis C and Maguire JD. Melioidosis masquerading as community-acquired pneumonia: a case report demonstrating efcacy of intrapleural brinolytic therapy. J travel Med. 2009 Jan-Feb;16(1):74-7.
