In-vitro activity of ß-lactams/trimethoprim-sulfamethoxazole combinations against different strains of Burkholderia pseudomallei.

Trimethoprim-sulfamethoxazole is an active agent against Burkholderia pseudomallei and is being used in intensive and maintenance phases of melioidosis therapy. In this study, we evaluated the bactericidal activities of ß-lactams (imipenem, ceftazidime and amoxicillin- clavulanate) alone and in combinations with trimethoprim-sulfamethoxazole against B. pseudomallei . Four clinical strains of B. pseudomallei were selected based on different genotypes that are frequently found in Malaysia. The minimum inhibitory concentrations of trimethoprim-sulfamethoxazole, ceftazidime, imipenem and amoxicillin-clavulanate were determined using microdilution broth method. The bactericidal activities and synergy effects of ß-lactams and/or trimethoprim-sulfamethoxazole were evaluated by checkerboard and static time-kill analyses at 1×MIC concentration of each antibiotic. Using checkerboard method, the ß-lactam/trimethoprim-sulfamethoxazole combinations exhibited ΣFIC of 0.75-4.00. In time-kill analysis, ceftazidime/trimethoprim-sulfamethoxazole combination demonstrated synergy against three strains (less 2.25-2.41 log 10 CFU/mL compared to the most active antibiotic monotherapy) whereas imipenem/trimethoprim-sulfamethoxazole combination regimen showed synergy against one strain (less 3.32 log 10 CFU/mL). No antagonist effect or major re-growth was observed in all combination regimens, whereas 11 out of 12 of ß-lactam monotherapy regimens were associated with re-growth of bacteria. However, all ß-lactam monotherapy regimens exhibited rapid and stronger killing activities against BUPS/07/14, in the initial 12 hours compared to ß-lactam/ trimethoprim- sulfamethoxazole combination regimens. The combination of ß-lactams with trimethoprim- sulfamethoxazole demonstrated better killing effect at 24 hours compared to monotherapy and no major bacterial regrowth was observed. Nevertheless, delay in killing activities of ß-lactam/trimethoprim-sulfamethoxazole combination regimens against BUPS/07/14 need further examination because this phenomenon can lead to treatment failure in some patients.

Identification and characterization of two drug-like fragments that bind to the same cryptic binding pocket of Burkholderia pseudomallei DsbA.

Disulfide-bond-forming proteins (Dsbs) play a crucial role in the pathogenicity of many Gram-negative bacteria. Disulfide-bond-forming protein A (DsbA) catalyzes the formation of the disulfide bonds necessary for the activity and stability of multiple substrate proteins, including many virulence factors. Hence, DsbA is an attractive target for the development of new drugs to combat bacterial infections. Here, two fragments, bromophenoxy propanamide (1) and 4-methoxy-N-phenylbenzenesulfonamide (2), were identified that bind to DsbA from the pathogenic bacterium Burkholderia pseudomallei, the causative agent of melioidosis. The crystal structures of oxidized B. pseudomallei DsbA (termed BpsDsbA) co-crystallized with 1 or 2 show that both fragments bind to a hydrophobic pocket that is formed by a change in the side-chain orientation of Tyr110. This conformational change opens a `cryptic' pocket that is not evident in the apoprotein structure. This binding location was supported by 2D-NMR studies, which identified a chemical shift perturbation of the Tyr110 backbone amide resonance of more than 0.05 p.p.m. upon the addition of 2 mM fragment 1 and of more than 0.04 p.p.m. upon the addition of 1 mM fragment 2. Although binding was detected by both X-ray crystallography and NMR, the binding affinity (Kd) for both fragments was low (above 2 mM), suggesting weak interactions with BpsDsbA. This conclusion is also supported by the crystal structure models, which ascribe partial occupancy to the ligands in the cryptic binding pocket. Small fragments such as 1 and 2 are not expected to have a high energetic binding affinity due to their relatively small surface area and the few functional groups that are available for intermolecular interactions. However, their simplicity makes them ideal for functionalization and optimization. The identification of the binding sites of 1 and 2 to BpsDsbA could provide a starting point for the development of more potent novel antimicrobial compounds that target DsbA and bacterial virulence.

Comparative clinical characteristics and outcomes of patients with community acquired bacteremia caused by Escherichia coli, Burkholderia pseudomallei and Staphylococcus aureus: A prospective observational study (Ubon-sepsis).

BACKGROUND: Community acquired bacteremia (CAB) is a common cause of sepsis in low and middle-income countries (LMICs). However, knowledge about factors associated with outcomes of CAB in LMICs is limited. METHODOLOGY/PRINCIPAL FINDINGS: A prospective observational study (Ubon-sepsis) of adults admitted to a referral hospital with community-acquired infection in Northeastern Thailand was conducted between March 1, 2013 and February 1, 2017. In the present analysis, patients with a blood culture collected within 24 hours of admission that was positive for one of the three most common pathogens were studied. Clinical features, management, and outcomes of patients with each cause of CAB were compared. Of 3,806 patients presenting with community-acquired sepsis, 155, 131 and 37 patients had a blood culture positive for Escherichia coli, Burkholderia pseudomallei and Staphylococcus aureus, respectively. Of these 323 CAB patients, 284 (89%) were transferred from other hospitals. 28-day mortality was highest in patients with B. pseudomallei bactaeremia (66%), followed by those with S. aureus bacteraemia (43%) and E. coli (19%) bacteraemia. In the multivariable Cox proportional hazards model adjusted for age, sex, transfer from another hospital, empirical antibiotics prior to or during the transfer, and presence of organ dysfunction on admission, B. pseudomallei (aHR 3.78; 95%CI 2.31-6.21) and S. aureus (aHR 2.72; 95%CI 1.40-5.28) bacteraemias were associated with higher mortality compared to E. coli bacteraemia. Receiving empirical antibiotics recommended for CAB caused by the etiologic organism prior to or during transfer was associated with survival (aHR 0.58; 95%CI 0.38-0.88). CONCLUSIONS/SIGNIFICANCE: Mortality of patients with CAB caused by B. pseudomallei was higher than those caused by S. aureus and E. coli, even after adjusting for presence of organ dysfunction on admission and effectiveness of empirical antibiotics received. Improving algorithms or rapid diagnostic tests to guide early empirical antibiotic may be key to improving CAB outcomes in LMICs.

Risk Factors for Mortality in Melioidosis: A Single-Centre, 10-Year Retrospective Cohort Study.

Melioidosis is a tropical infectious disease with diverse clinical presentations. We aimed to investigate the characteristics and mortality risk factors of patients diagnosed with melioidosis in the past 10 years. This was a retrospective cohort study conducted at a quaternary care centre in South India. Clinical, demographic, and biochemical data in patients diagnosed with melioidosis with cultures were collected between January 2011 and December 2020 from medical records. Logistic regression analysis was performed to screen mortality risk factors of melioidosis in addition to descriptive statistics and chi-square analysis. Seventy-three melioidosis patients' records were analysed, and the most common comorbidity was type 2 diabetes mellitus (n = 53, 72.6%). The patients showed diverse presentations: pulmonary involvement, 30 (41.1%); splenomegaly, 29 (39.7%); abscesses and cutaneous involvement, 18 (24.7%); lymph node, 10 (13.7%); arthritis and osteomyelitis, 9 (12.3%); and genitourinary infection, 4 (5.4%). The mortality was noted to be 15 (20.5%). Logistic regression analysis indicated that chronic kidney disease (OR = 14.0), CRP >100 IU/L (OR = 6.964), and S. albumin <3 gm/dl (OR = 8.0) were risk factors associated with mortality and can guide in risk stratification. Hypoalbuminemia is a novel mortality risk factor, detected in this study, and requires further investigation to validate its utility as a prognostic marker and reveal possible therapeutic benefits in clinical correction.

Tauroursodeoxycholic acid prevents Burkholderia pseudomallei-induced endoplasmic reticulum stress and is protective during melioidosis in mice.

BACKGROUND: Burkholderia pseudomallei, a facultative intracellular bacterium, is the aetiological agent of melioidosis that is responsible for up to 40% sepsis-related mortality in epidemic areas. However, no effective vaccine is available currently, and the drug resistance is also a major problem in the treatment of melioidosis. Therefore, finding new clinical treatment strategies in melioidosis is extremely urgent. RESULTS: We demonstrated that tauroursodeoxycholic acid (TUDCA), a clinically available endoplasmic reticulum (ER) stress inhibitor, can promote B. pseudomallei clearance both in vivo and in vitro. In this study, we investigated the effects of TUDCA on the survival of melioidosis mice, and found that treatment with TUDCA significantly decreased intracellular survival of B. pseudomallei. Mechanistically, we found that B. pseudomallei induced apoptosis and activated IRE1 and PERK signaling ways of ER stress in RAW264.7 macrophages. TUDCA treatment could reduce B. pseudomallei-induced ER stress in vitro, and TUDCA is protective in vivo. CONCLUSION: Taken together, our study has demonstrated that B. pseudomallei infection results in ER stress-induced apoptosis, and TUDCA enhances the clearance of B. pseudomallei by inhibiting ER stress-induced apoptosis both in vivo and in vitro, suggesting that TUDCA could be used as a potentially alternative treatment for melioidosis.

Drug screening to identify compounds to act as co-therapies for the treatment of Burkholderia species.

Burkholderia pseudomallei is a soil-dwelling organism present throughout the tropics. It is the causative agent of melioidosis, a disease that is believed to kill 89,000 people per year. It is naturally resistant to many antibiotics, requiring at least two weeks of intravenous treatment with ceftazidime, imipenem or meropenem followed by 6 months of orally delivered co-trimoxazole. This places a large treatment burden on the predominantly middle-income nations where the majority of disease occurs. We have established a high-throughput assay for compounds that could be used as a co-therapy to potentiate the effect of ceftazidime, using the related non-pathogenic bacterium Burkholderia thailandensis as a surrogate. Optimization of the assay gave a Z' factor of 0.68. We screened a library of 61,250 compounds and identified 29 compounds with a pIC50 (-log10(IC50)) greater than five. Detailed investigation allowed us to down select to six "best in class" compounds, which included the licensed drug chloroxine. Co-treatment of B. thailandensis with ceftazidime and chloroxine reduced culturable cell numbers by two orders of magnitude over 48 hours, compared to treatment with ceftazidime alone. Hit expansion around chloroxine was performed using commercially available compounds. Minor modifications to the structure abolished activity, suggesting that chloroxine likely acts against a specific target. Finally, an initial study demonstrates the utility of chloroxine to act as a co-therapy to potentiate the effect of ceftazidime against B. pseudomallei. This approach successfully identified potential co-therapies for a recalcitrant Gram-negative bacterial species. Our assay could be used more widely to aid in chemotherapy to treat infections caused by these bacteria.

Tannic Acid-Stabilized Silver Nanoparticles Used in Biomedical Application as an Effective Antimelioidosis and Prolonged Efflux Pump Inhibitor against Melioidosis Causative Pathogen.

Burkholderia pseudomallei is the causative pathogen of melioidosis and this bacterium is resistant to several antibiotics. Silver nanoparticles (AgNPs) are an interesting agent to develop to solve this bacterial resistance. Here, we characterize and assess the antimelioidosis activity of AgNPs against these pathogenic bacteria. AgNPs were characterized and displayed a maximum absorption band at 420 nm with a spherical shape, being well-monodispersed and having high stability in solution. The average size of AgNPs is 7.99 ± 1.46 nm. The antibacterial efficacy of AgNPs was evaluated by broth microdilution. The bactericidal effect of AgNPs was further assessed by time-kill kinetics assay. Moreover, the effect of AgNPs on the inhibition of the established biofilm was investigated by the crystal violet method. In parallel, a study of the resistance induction development of B. pseudomallei towards AgNPs with efflux pump inhibiting effect was performed. We first found that AgNPs had strong antibacterial activity against both susceptible and ceftazidime-resistant (CAZ-resistant) strains, as well as being efficiently active against B. pseudomallei CAZ-resistant strains with a fast-killing mode via a bactericidal effect within 30 min. These AgNPs did not only kill planktonic bacteria in broth conditions, but also in established biofilm. Our findings first documented that the resistance development was not induced in B. pseudomallei toward AgNPs in the 30th passage. We found that AgNPs still showed an effective efflux pump inhibiting effect against these bacteria after prolonged exposure to AgNPs at sublethal concentrations. Thus, AgNPs have valuable properties for being a potent antimicrobial agent to solve the antibiotic resistance problem in pathogens.

A study of inhibitors of d-glycero-ß-d-manno-heptose-1-phosphate adenylyltransferase from Burkholderia pseudomallei as a potential antibiotic target.

d-Glycero-ß-d-manno-heptose-1-phosphate adenylyltransferase from Burkholderia pseudomallei (BpHldC) is the fourth enzyme in the ADP-l-glycero-ß-d-manno-heptose biosynthesis pathway producing a lipopolysaccharide core. Therefore, BpHldC is an anti-melioidosis target. Three ChemBridge compounds purchased from ChemBridge Corporation (San Diego, CA) were found to have an effective inhibitory activity on BpHldC. Interestingly, ChemBridge 7929959 was the most effective compound due to the presence of the terminal benzyl group. The enzyme kinetic study revealed that most of them show mixed type inhibitory modes against ATP and ßG1P. The induced-fit docking indicated that the medium affinity of ChemBridge 7929959 is originated from its benzyl group occupying the substrate-binding pocket of BpHldC. The inhibitory role of terminal aromatic groups was proven with ChemBridge 7570508. Combined with the previous study, ChemBridge 7929959 is found to work as a dual inhibitor against both HldC and HddC. Therefore, three ChemBridge compounds can be developed as a potent anti-melioidosis agent with a novel inhibitory concept.

Identification and Characterization of Burkholderia pseudomallei from Localized Pyogenic Infections in Eastern India: A Clinico-Microbiological Study.

Melioidosis, caused by Burkholderia pseudomallei, is increasingly recognized in several regions of the globe. The present study was performed to identify and determine the frequency of B. pseudomallei infection in localized pyogenic lesions in eastern India and describe their clinico-microbiological profile. Pus samples were subjected to standard microbiological techniques for isolation and identification of various bacteria, including B. pseudomallei, which were confirmed by PCR. The clinical and demographic details of patients with melioidosis and antimicrobial susceptibility pattern of B. pseudomallei isolates were analyzed. Of 245 samples, 126 (51.4%) were culture positive, yielding 137 isolates. Staphylococcus aureus was the predominant pathogen accounting for 54 (39.4%) isolates, followed by B. pseudomallei accounting for 34 (24.8%) isolates. The mean age of the patients with melioidosis was 39.1 years, with males (24/34; 70.6%) being affected more than females (10/34; 29.4%). A majority of the patients were laborers (12/34; 35.3), followed by homemakers (8/34; 23.5%). Head and neck abscesses (35.3%) were the most common presentation followed by pyogenic lesions of the musculoskeletal system (32.3%) and deep organ abscesses (23.5%). Clinical resolution of infection was observed in 31 (91.2%) patients, relapse in two (5.9%) patients, and death in one (2.9%) patient, respectively. Susceptibility testing revealed all B. pseudomallei isolates to be completely susceptible to the following antimicrobials: ceftazidime, trimethoprim-sulfamethoxazole, imipenem, and doxycycline, with one (2.9%) resistant to amoxicillin-clavulanic acid. Burkholderia pseudomallei is an emerging etiological agent of localized pyogenic infections in eastern India, affecting a mainly adult male population. An increased vigilance along with appropriate diagnostic techniques helps in accurate diagnosis facilitating appropriate therapy.

A triple-targeting inhibitory activity of Rose Bengal on polysaccharide biosynthesis of Burkholderia pseudomallei.

Sugar nucleotidyltransferases (SNTs) participate in various biosynthesis pathways constructing polysaccharides in Gram-negative bacteria. In this study, a triple-targeting inhibitory activity of Rose Bengal against SNTs such as d-glycero-α-d-manno-heptose-1-phosphate guanylyltransferase (HddC), d-glycero-ß-d-manno-heptose-1-phosphate adenylyltransferase (HldC), and 3-deoxy-d-manno-oct-2-ulosonic acid cytidylyltransferase (KdsB) from Burkholderia pseudomallei is provided. Rose Bengal effectively suppresses the nucleotidyltransferase activity of the three SNTs, and its IC50 values are 10.42, 0.76, and 5.31 µM, respectively. Interestingly, Rose Bengal inhibits the three enzymes regardless of their primary, secondary, tertiary, and quaternary structural differences. The experimental results indicate that Rose Bengal possesses the plasticity to shape its conformation suitable to interact with the three SNTs. As HddC functions in the formation of capsular polysaccharides and HldC and KdsB produce building blocks to constitute the inner core of lipopolysaccharide, Rose Bengal is a potential candidate to design antibiotics in a new category. In particular, it can be developed as a specific antimelioidosis agent. As the mortality rate of the infected people caused by B. pseudomallei is quite high, there is an urgent need for specific antimelioidosis agents. Therefore, a further study is being carried out with derivatives of Rose Bengal.

Antimicrobial Susceptibility of Western Hemisphere Isolates of Burkholderia pseudomallei: Phenotypic and Genomic Analyses.

Current antimicrobial treatment recommendations for melioidosis, the disease caused by Burkholderia pseudomallei, are largely based on studies of strains isolated from the Eastern Hemisphere (EH), where most human cases are identified and reported. In this study, we evaluated the antimicrobial susceptibility of 26 strains in the CDC (Centers for Diseases Control and Prevention) collection from the Western Hemisphere (WH) isolated from 1960 to 2015. Minimal inhibitory concentration (MIC) values were measured by standard broth microdilution for 16 antimicrobials following Clinical and Laboratory Standards Institute (CLSI) guidelines. Twenty-four of the 26 WH strains were susceptible to the six antimicrobials with CLSI-defined MIC susceptibility interpretive criteria for B. pseudomallei: amoxicillin/clavulanate, ceftazidime, imipenem, doxycycline, tetracycline, and trimethoprim/sulfamethoxazole. One WH strain demonstrated intermediate amoxicillin/clavulanate resistance and another strain had intermediate resistance to tetracycline. For all antimicrobials tested, the susceptibility profiles of WH isolates were comparable with previously reported MIC results of EH strains. The overall similarities suggest that the same antimicrobials are useful for melioidosis treatment in both the WH and EH. Using in silico analyses of WH genomes, we identified a novel amino acid substitution P258S in the beta-lactamase PenA, which may contribute to decreased susceptibility to amoxicillin/clavulanate in B. pseudomallei.

Genomic and RT-qPCR analysis of trimethoprim-sulfamethoxazole and meropenem resistance in Burkholderia pseudomallei clinical isolates.

BACKGROUND: Melioidosis is an endemic disease in southeast Asia and northern Australia caused by the saprophytic bacteria Burkholderia pseudomallei, with a high mortality rate. The clinical presentation is multifaceted, with symptoms ranging from acute septicemia to multiple chronic abscesses. Here, we report a chronic case of melioidosis in a patient who lived in Malaysia in the 70s and was suspected of contracting tuberculosis. Approximately 40 years later, in 2014, he was diagnosed with pauci-symptomatic melioidosis during a routine examination. Four strains were isolated from a single sample. They showed divergent morphotypes and divergent antibiotic susceptibility, with some strains showing resistance to trimethoprim-sulfamethoxazole and fluoroquinolones. In 2016, clinical samples were still positive for B. pseudomallei, and only one type of strain, showing atypical resistance to meropenem, was isolated. PRINCIPAL FINDINGS: We performed whole genome sequencing and RT-qPCR analysis on the strains isolated during this study to gain further insights into their differences. We thus identified two types of resistance mechanisms in these clinical strains. The first one was an adaptive and transient mechanism that disappeared during the course of laboratory sub-cultures; the second was a mutation in the efflux pump regulator amrR, associated with the overexpression of the related transporter. CONCLUSION: The development of such mechanisms may have a clinical impact on antibiotic treatment. Indeed, their transient nature could lead to an undiagnosed resistance. Efflux overexpression due to mutation leads to an important multiple resistance, reducing the effectiveness of antibiotics during treatment.

Taking the next-gen step: Comprehensive antimicrobial resistance detection from Burkholderia pseudomallei.

BACKGROUND: Antimicrobial resistance (AMR) poses a major threat to human health. Whole-genome sequencing holds great potential for AMR identification; however, there remain major gaps in accurately and comprehensively detecting AMR across the spectrum of AMR-conferring determinants and pathogens. METHODS: Using 16 wild-type Burkholderia pseudomallei and 25 with acquired AMR, we first assessed the performance of existing AMR software (ARIBA, CARD, ResFinder, and AMRFinderPlus) for detecting clinically relevant AMR in this pathogen. B. pseudomallei was chosen due to limited treatment options, high fatality rate, and AMR caused exclusively by chromosomal mutation (i.e. single-nucleotide polymorphisms [SNPs], insertions-deletions [indels], copy-number variations [CNVs], inversions, and functional gene loss). Due to poor performance with existing tools, we developed ARDaP (Antimicrobial Resistance Detection and Prediction) to identify the spectrum of AMR-conferring determinants in B. pseudomallei. FINDINGS: CARD, ResFinder, and AMRFinderPlus failed to identify any clinically-relevant AMR in B. pseudomallei; ARIBA identified AMR encoded by SNPs and indels that were manually added to its database. However, none of these tools identified CNV, inversion, or gene loss determinants, and ARIBA could not differentiate AMR determinants from natural genetic variation. In contrast, ARDaP accurately detected all SNP, indel, CNV, inversion, and gene loss AMR determinants described in B. pseudomallei (n≈50). Additionally, ARDaP accurately predicted three previously undescribed determinants. In mixed strain data, ARDaP identified AMR to as low as ~5% allelic frequency. INTERPRETATION: Existing AMR software packages are inadequate for chromosomal AMR detection due to an inability to detect resistance conferred by CNVs, inversions, and functional gene loss. ARDaP overcomes these major shortcomings. Further, ARDaP enables AMR prediction from mixed sequence data down to 5% allelic frequency, and can differentiate natural genetic variation from AMR determinants. ARDaP databases can be constructed for any microbial species of interest for comprehensive AMR detection. FUNDING: National Health and Medical Research Council (BJC, EPP, DSS); Australian Government (DEM, ES); Advance Queensland (EPP, DSS).

Predicting toxins found in toxin-antitoxin systems with a role in host-induced Burkholderia pseudomallei persistence.

Burkholderia pseudomallei (Bpm) is a bacterial pathogen that causes Melioidosis, a disease with up to 40% mortality and an infection relapse of 15-23% despite antibiotic treatment. Ineffective clearance of Bpm by antibiotics is believed to be due to persistence, a hibernation-like survival mechanism modulated, in part, by toxin-antitoxin systems (TAS). Several organisms possess a repertoire of TASs but defining environmental cues eliciting their activity is hindered by laborious in vitro experiments, especially when there are many toxins with redundant function. Here, we identified which of 103 proteins in Bpm that share features found in toxins of the TAS and repurposed transcriptional data to identify which ones play a role in surviving intracellular host defenses. Putative toxins with the strongest transcriptional response were found to have low conservation between Bpm strains, while toxins that were constitutively expressed were highly conserved. Further examination of highly conserved toxins BPSS0899, BPSS1321, and BPSL1494 showed that they were functional, and their mutation led to reduce survival within macrophages and reduced in vivo persistence-associated pathology (abscesses) during treatment, but did not affect macrophages persistence. These findings highlight the utility of a data-driven approach to select putative toxins and suggests a selective role for some TAS in host survival.

Rhamnolipid enhances Burkholderia pseudomallei biofilm susceptibility, disassembly and production of virulence factors.

Aim: This study evaluated the effect of the biosurfactant rhamnolipid on the antimicrobial susceptibility, biofilm growth dynamics and production of virulence factors by Burkholderia pseudomallei. Materials & methods: The effects of rhamnolipid on planktonic and biofilm growth and its interaction with antibacterial drugs were evaluated. Then, its effects on growing and mature biofilms and on protease and siderophore production were assessed. Results: Rhamnolipid did not inhibit B. pseudomallei growth, but significantly enhanced the activity of meropenem and amoxicillin-clavulanate against mature biofilms. Rhamnolipid significantly reduced the biomass of mature biofilms, significantly increased protease production by growing and mature biofilms and siderophore release by growing biofilms. Conclusion: Rhamnolipid enhances the antimicrobial activity against B. pseudomallei, assists biofilm disassembly and alters protease and siderophore production by bacterial biofilms.

The role of chemoenzymatic synthesis in advancing trehalose analogues as tools for combatting bacterial pathogens.

Trehalose, a disaccharide of glucose, is increasingly recognized as an important contributor to virulence in major bacterial pathogens, such as Mycobacterium tuberculosis, Clostridioides difficile, and Burkholderia pseudomallei. Accordingly, bacterial trehalose metabolic pathways that are not present in humans have gained traction as targets for antibiotic and diagnostic development. Toward this goal, trehalose can be modified through a combination of rational design and synthesis to produce functionalized trehalose analogues, which can be deployed to probe or inhibit bacterial trehalose metabolism. However, the unique α,α-1,1-glycosidic bond and C2 symmetry of trehalose make analogue synthesis via traditional chemical methods very challenging. We and others have turned to the creation of chemoenzymatic synthesis methods, which in principle allow the use of nature's trehalose-synthesizing enzymes to stereo- and regioselectively couple simple, unprotected substrates to efficiently and conveniently generate trehalose analogues. Here, we provide a contextual account of our team's development of a trehalose analogue synthesis method that employs a highly substrate-tolerant, thermostable trehalose synthase enzyme, TreT from Thermoproteus tenax. Then, in three vignettes, we highlight how chemoenzymatic synthesis has accelerated the development of trehalose-based imaging probes and inhibitors that target trehalose-utilizing bacterial pathogens. We describe the role of TreT catalysis and related methods in the development of (i) tools for in vitro and in vivo imaging of mycobacteria, (ii) anti-biofilm compounds that sensitize drug-tolerant mycobacteria to clinical anti-tubercular compounds, and (iii) degradation-resistant trehalose analogues that block trehalose metabolism in C. difficile and potentially other trehalose-utilizing bacteria. We conclude by recapping progress and discussing priorities for future research in this area, including improving the scope and scale of chemoenzymatic synthesis methods to support translational research and expanding the functionality and applicability of trehalose analogues to study and target diverse bacterial pathogens.

Mycotic aneurysm secondary to melioidosis in China: A series of eight cases and a review of literature.

Burkholderia pseudomallei is the causative agent of melioidosis, endemic in Southeast Asia and Northern Australia, and increasingly recognized in southern China, especially in Hainan Province. Mycotic aneurysm caused by B. pseudomallei is a rare but potentially severe illness with a high mortality rate. The clinical features of the mycotic aneurysm secondary to melioidosis have not been illustrated in China. Over a seven-year period (2013 to 2019), 159 patients with bacteremic melioidosis were retrospectively analyzed in Hainan province, China, of whom eight patients were confirmed to have mycotic aneurysm through the combination of imaging examination, pathologic examination and aneurysm tissue culture. We summarized these eight patients' clinical characteristics, demographical features, treatments and outcomes. The susceptibilities to five commonly-used antibiotics for these eight B. pseudomallei isolates were also determined by E-test strips. Furthermore, the mycotic aneurysm cases secondary to melioidosis retrieved from the literature were also reviewed. Of the eight cases, six had abdominal mycotic aneurysms, one had a left iliac aneurysm, and the other one had an infectious mesenteric aneurysm. They were aged from 48 to 69 years old, and had the underlying risk factors of diabetes mellitus (2 patients), long-term smoking (4 patients), hypertension (6 patients), and soil and water contact history (6 patients), respectively. The positive arterial aneurysm imaging was observed in all patients via computed tomography (CT) or angiography. Eight B. pseudomallei isolates collected from both blood and mycotic aneurysm tissues remained 100% susceptible to imipenem and ceftazidime. After surgery combined with antibiotic administration, six patients survived, with a mortality rate of 25%. In melioidosis endemic areas, the mycotic aneurysm secondary to melioidosis might be underdiagnosed, and increased awareness of predisposing risk factors and clinical features of the mycotic aneurysm is required. Following a positive B. pseudomallei blood culture, the diagnosis of mycotic aneurysm should be under consideration in those with abdominal pain and/or hypertension. Imaging by CT or angiography is indispensable for its timely diagnosis and management.

Optical microscopy reveals the dynamic nature of B. pseudomallei morphology during ß-lactam antimicrobial susceptibility testing.

BACKGROUND: In Gram-negative species, ß-lactam antibiotics target penicillin binding proteins (PBPs) resulting in morphological alterations of bacterial cells. Observations of antibiotic-induced cell morphology changes can rapidly and accurately differentiate drug susceptible from resistant bacterial strains; however, resistant cells do not always remain unchanged. Burkholderia pseudomallei is a Gram-negative, biothreat pathogen and the causative agent of melioidosis, an often fatal infectious disease for humans. RESULTS: Here, we identified ß-lactam targets in B. pseudomallei by in silico analysis. Ten genes encoding putative PBPs, including PBP-1, PBP-2, PBP-3 and PBP-6, were detected in the genomes of susceptible and resistant strains. Real-time, live-cell imaging of B. pseudomallei strains demonstrated dynamic morphological changes in broth containing clinically relevant ß-lactam antibiotics. At sub-inhibitory concentrations of ceftazidime (CAZ), amoxicillin-clavulanic acid (AMC), and imipenem (IPM), filamentation, varying in length and proportion, was an initial response of the multidrug-resistant strain Bp1651 in exponential phase. However, a dominant morphotype reemerged during stationary phase that resembled cells unexposed to antibiotics. Similar morphology dynamics were observed for AMC-resistant strains, MSHR1655 and 724644, when exposed to sub-inhibitory concentrations of AMC. For all B. pseudomallei strains evaluated, increased exposure time and exposure to increased concentrations of AMC at and above minimal inhibitory concentrations (MICs) in broth resulted in cell morphology shifts from filaments to spheroplasts and/or cell lysis. B. pseudomallei morphology changes were more consistent in IPM. Spheroplast formation followed by cell lysis was observed for all strains in broth containing IPM at concentrations greater than or equal to MICs, however, the time to cell lysis was variable. B. pseudomallei cell lengths were strain-, drug- and drug concentration-dependent. CONCLUSIONS: Both resistant and susceptible B. pseudomallei strains exhibited filamentation during early exposure to AMC and CAZ at concentrations used to interpret susceptibility (based on CLSI guidelines). While developing a rapid ß-lactam antimicrobial susceptibility test based on cell-shape alone requires more extensive analyses, optical microscopy detected B. pseudomallei growth attributes that lend insight into antibiotic response and antibacterial mechanisms of action.

Revisiting aminocoumarins for the treatment of melioidosis.

Burkholderia pseudomallei causes melioidosis, a potentially lethal disease that can establish both chronic and acute infections in humans. It is inherently recalcitrant to many antibiotics, there is a paucity of effective treatment options and there is no vaccine. In the present study, the efficacies of selected aminocoumarin compounds, DNA gyrase inhibitors that were discovered in the 1950s but are not in clinical use for the treatment of melioidosis were investigated. Clorobiocin and coumermycin were shown to be particularly effective in treating B. pseudomallei infection in vivo. A novel formulation with dl-tryptophan or l-tyrosine was shown to further enhance aminocoumarin potency in vivo. It was demonstrated that coumermycin has superior pharmacokinetic properties compared with novobiocin, and the coumermycin in l-tyrosine formulation can be used as an effective treatment for acute respiratory melioidosis in a murine model. Repurposing of existing approved antibiotics offers new resources in a challenging era of drug development and antimicrobial resistance.