BackgroundIsolation of the soil bacterium Burkholderia pseudomallei from tropical environments is important to generate a global risk map for man and animals to acquire the infectious disease melioidosis. There is increasing evidence, that the currently recommended soil culture protocol using threonine-basal salt solution with colistin (TBSS-C50) for enrichment of B. pseudomallei and Ashdown agar for subsequent subculture lacks sensitivity. We therefore investigated, if the otherwise rarely encountered erythritol catabolism of B. pseudomallei might be exploited to improve isolation of this bacterium from soil.Methodology/Principal findingsBased on TBSS-C50, we designed a new colistin-containing medium with erythritol as the single carbon source (EM). This medium was validated in various culture protocols by analyzing 80 soil samples from 16 different rice fields in Vietnam. B. pseudomallei enrichment was determined in all culture supernatants by a specific quantitative PCR (qPCR) targeting the type three secretion system 1. 51 out of 80 (63.8%) soil samples gave a positive qPCR signal in at least one of the culture conditions. We observed a significantly higher enrichment shown by lower median cycle threshold values for B. pseudomallei in a two-step culture with TBSS-C50 for 48 h followed by EM for 96h compared to single cultures in TBSS-C50 for either 48h or 144h (p<0.0001, respectively). Accordingly, B. pseudomallei could be isolated on Ashdown agar in 58.8% (30/51) of samples after subcultures from our novel two-step enrichment culture compared to only 9.8% (5/51) after standard enrichment with TBSS-C50 for 48h (p<0.0001) or 25.5% (13/51; p<0.01) after TBSS-C50 for 144h.Conclusions/significanceIn the present study, we show that specific exploitation of B. pseudomallei metabolic capabilities in enrichment protocols leads to a significantly improved isolation rate of this pathogen from soil compared to established standard procedures. Our new culture method might help to facilitate the creation of environmental risk maps for melioidosis in the future.

T he gram-negative soil-dwelling saprophytic bacterium Burkholderia pseudomallei causes melioidosis, a fatal disease highly endemic to Southeast Asia and northern Australia (1). Humans can be infected with B. pseudomallei via inoculation, inhalation, and ingestion. Rice farmers are at high risk for infection because of their frequent exposure to soil and water, but newborns, children, and older persons also are at risk (2,3). We report 3 melioidosis deaths among children in northern Vietnam. The StudyIn November 2019, the Preventive Health Center of Soc Son district in Vietnam reported the deaths of 3 children from 1 family. The first child, a 7-year-old girl, had a high fever and abdominal pain on April 6, 2019. Two days later, she was admitted to a local hospital; after 1 day, she was transferred to St. Paul Hospital in Hanoi, where septic shock was diagnosed. She died on April 9, shortly after admission, before any diagnostic tests were performed.On October 27, 2019, the second child, a 5-yearold boy, had a high fever and abdominal pain around the umbilicus. He was admitted to Vietnam National Children's Hospital in Hanoi on October 28 with diagnosed septic shock. Abdominal and chest radiographs and abdominal ultrasound results were unremarkable. His blood culture grew B. pseudomallei, and he died on October 31.The third child, a 13-month-old boy, had a high fever and poor appetite on November 10, 2019. According to his grandparents, he had black stool, like his sister and brother. He was admitted to Vietnam National Children's Hospital; chest radiography results were unremarkable, but B. pseudomallei was cultured from his blood sample. He died on November 16.We retrieved laboratory findings from all hospitals to which these children were admitted. Results showed leukopenia, neutropenia, thrombocytopenia, and high procalcitonin and C-reactive protein in all children's blood. Liver dysfunction was diagnosed in all 3 children, but kidney dysfunction was recognized only in the 2 older children. We detected no identifiable risk factors (Table 1).To trace the source of infection, on November 17, 2019, we visited the family home in the midland region of northern Vietnam (Figure 1). During our active surveillance for melioidosis cases admitted to provincial and tertiary hospitals surrounding Hanoi (4), no previous cases had been reported from this area.We interviewed the parents and grandparents using epidemiologic questions about all the children's daily activities inside and outside the house. The family used water supplied from 3 boreholes: 1 for bathing (borehole A), 1 for livestock (borehole B), and 1 for human consumption (borehole C). During our first environmental investigation, we collected samples of front garden soil (n = 7), borehole water (n = 9), and boiled drinking water (n = 1). We performed qualitative culture for B. pseudomallei, and all 3 water samples collected from borehole A tested positive (Appendix, https://wwwnc.cdc.gov/EID/ article/28/8/22-0113-App1.pdf).

Melioidosis is a fatal infectious disease in the tropics and subtropics. Currently, bacterial culture is the gold standard for diagnosis of the disease, but its sensitivity is relatively low. In this study, we evaluated four ELISAs using sera collected from culture-confirmed cases of melioidosis (n = 63), cases with other bacterial infections (n = 62), and healthy donors (n = 60). Antigens used for ELISAs were the whole-cell (WC) antigens and recombinant proteins of hemolysis co-regulated protein 1 (Hcp1), GroEL1, and alkyl hydroperoxide reductase subunit C (AhpC). Using the cutoff values for optical density at 490 nm defined at a specificity of > 95%, the sensitivity of the WC, Hcp1, GroEL1, and AhpC ELISAs was 93.7%, 87.3%, 61.9%, and 57.1%, respectively. The combined WC/Hcp1 ELISA showed the greatest sensitivity and specificity of 98.4% and 95.1%, respectively. Of 511 and 500 sera collected from clinically suspected febrile patients admitted to the General Hospital of Ha Tinh Province and the Hue Central Hospital, respectively, combined WC/Hcp1 ELISAs showed 52 (10.2%) and 41 (8.2%) patients positive for melioidosis, respectively. The assay detected 14 of 14 (100%) and 21 of 23 (91.3%) culture-confirmed cases of melioidosis at Ha Tinh and Hue, respectively. A follow-up study of 38 patients positive for melioidosis by combined WC/Hcp1 ELISAs but negative for Burkholderia pseudomallei by culture method or not assigned to examine for bacterial culture resulted in 2 (5.3%) culture-reconfirmed patients with melioidosis, 9 (23.7%) deaths, 17 (44.7%) unhealthy patients, and 10 (26.3%) healthy persons. Combined WC/Hcp1 ELISA was a reliable serological method to detect underdiagnosed cases of melioidosis. Further investigations are needed to estimate the true sensitivity and specificity of the assay and the true number of cases of melioidosis.

Background Burkholderia pseudomallei is a causative agent of melioidosis, a fatal infectious disease highly prevalent in the tropics where traditional medicinal plants are widely used for the treatment of various human ailments. In this study, we aimed to evaluate the in vitro antibacterial activity of common eligible herbs and medicinal plants against B. pseudomallei. Thermal and gastric stability, antibacterial spectrum, bactericidal activity, and cell cytotoxicity were also tested to verify the possible usage of these plants in the treatment of melioidosis. Results Eighteen eligible herbs and twenty-one medicinal plants were collected. Herb juices and aqueous plant samples extracted at different temperatures were prepared for antibacterial testing. A higher proportion of aqueous plant extracts (17/21; 80.9%) against B. pseudomallei was observed, in comparison with that of herb juices (8/18; 44.5%). Two herb juices and twelve aqueous plant extracts were selected for further tests. The juices of A. sativum and A. tuberosum decreased their antimicrobial activity when treated at higher temperatures whereas the aqueous plant extracts increased their antimicrobial activity when prepared at 70 and 100 °C. The herb juices showed a broader spectrum of antimicrobial activity than the aqueous plant extracts. All samples showed less cytotoxicity on the HT29, HepG2, and HEK293 cell lines. At the 2× minimal inhibitory concentration (MIC), aqueous extracts of Blechnum orientale, Breynia fruticose, Psidium guajava, Rhodomyrtus tomentosa, Rosa odorata, and Schima wallichii showed similar bactericidal activity to that of amoxicillin clavulanic acid. The antimicrobial activity of Mangifera indica, Punica granatum, and R. tomentosa remained under the stimulated gastric conditions. Conclusion Our data indicate that traditional medicinal plants prepared by decoction could be effectively used to treat melioidosis via the oral route. Further in vivo investigations are needed to explore other alternative therapies for the prevention and treatment of tested pathogenic bacterial species.