The essential functions of a bacterial pathogen reflect the most basic processes required for its viability and growth, and represent potential therapeutic targets. Most screens for essential genes have assayed a single condition-growth in a rich undefined medium-and thus have not distinguished genes that are generally essential from those that are specific to this particular condition. To help define these classes for Pseudomonas aeruginosa, we identified genes required for growth on six different media, including a medium made from cystic fibrosis patient sputum. The analysis used the Tn-seq circle method to achieve high genome coverage and analyzed more than 1,000,000 unique insertion positions (an average of one insertion every 6.0 bp). We identified 352 general and 199 condition-specific essential genes. A subset of assignments was verified in individual strains with regulated expression alleles. The profile of essential genes revealed that, compared with Escherichia coli, P. aeruginosa is highly vulnerable to mutations disrupting central carbon-energy metabolism and reactive oxygen defenses. These vulnerabilities may arise from the stripped-down architecture of the organism's carbohydrate utilization pathways and its reliance on respiration for energy generation. The essential function profile thus provides fundamental insights into P. aeruginosa physiology as well as identifying candidate targets for new antibacterial agents.Tn-seq | ESKAPE | cystic fibrosis | antibiotic target | sputum

Burkholderia pseudomallei is the causative agent of melioidosis, an overwhelming, rapidly fatal septic infection, and B. thailandensis is a closely related, less virulent species. Both organisms are naturally competent for DNA transformation, and this report describes a procedure exploiting this property for the rapid generation of marked deletion mutations by using PCR products. The method was employed to create 61 mutant strains. Several selectable elements were employed, including elements carrying loxP and FRT recombinase recognition sites to facilitate resistance marker excision. Chromosomal mutations could also be transferred readily between strains by transformation. The availability of simple procedures for creating defined chromosomal mutations and moving them between strains should facilitate genetic analysis of virulence and other traits of these two Burkholderia species.Burkholderia pseudomallei and Burkholderia thailandensis are closely related gram-negative bacteria widely distributed in soils of Southeast Asia and northern Australia (19,21). B. pseudomallei is the causative agent of melioidosis, whereas B. thailandensis is rarely associated with human disease but can kill rodents at high infectious doses (14,22). The B. thailandensis genome is smaller than that of B. pseudomallei (6.7 versus 7.2 Mbp) but encodes homologues of many of the established B. pseudomallei virulence determinants, including type III secretion systems and functions responsible for cellto-cell spread during infection (15,16,20). Phylogenomic comparisons imply that the two species share numerous additional traits as well (10, 23). Thus, B. thailandensis serves as a lowvirulence surrogate for studying numerous physiological and pathogenic characteristics of B. pseudomallei.Progress in the genetic analysis of B. thailandensis and B. pseudomallei has been limited by the lack of a general procedure for creating targeted mutations based on the genome sequences. Standard two-step plasmid-based procedures (18) employing sacB as a counterselective marker have not been generally successful for the two species due to the presence of endogenous sacB genes (although there have been exceptions) (6, 12). A simpler alternative, the direct generation of predefined mutations by transformation of PCR fragments, has greatly facilitated the genetic analysis of several bacterial species (7,11,17). In general, the fragments carry selectable markers flanked by regions of homology oriented such that homologous recombination replaces genomic sequences with the selectable marker. In this study, we developed such a procedure for B. thailandensis and B. pseudomallei which exploits the discovery, presented here, that these bacteria can be rendered naturally competent for DNA transformation. MATERIALS AND METHODS Bacterial strains and growth conditions. B. thailandensis E264 (from DonWoods, University of Calgary) and S95019 (Siriraj Hospital collection) and B. pseudomallei 1026a and 1026b (from Sharon Peacock, Wellcome Trust Unit, Bangkok, Thailand) were ...

In Thailand, leptospirosis is primarily associated with those who work in agricultural occupations. Leptospirosis control is hampered by a poor understanding of the complex interactions between humans, animal reservoirs, Leptospira, and the variable spatial environment in which these factors coexist. We aimed to address key knowledge gaps concerning leptospirosis disease dynamics and the human–animal–water-source interface in two high-risk areas in Thailand. We conducted a cross-sectional survey among 746 study participants in two high-risk areas for leptospirosis in Thailand: Sisaket (SSK) and Nakhon Si Thammarat (NST). Interactions among humans, animals and water sources were quantified and analyzed. The presence of different animal species and thus contact patterns were different in NST and SSK. The consumption of water from the shared sources between the two areas was different. Those whose occupations were related to animals or environmental water and those who consumed water from more than two sources were more likely to have been infected with leptospirosis, with adjusted odds ratios 4.31 (95% CI 1.17–15.83) and 10.74 (95% CI 2.28–50.53), respectively. Understanding specific water-source sharing networks and human–animal contact patterns is useful when designing national and area-specific control programmes to prevent and control leptospirosis outbreaks.