PCR combined with lateral flow dipstick assay (PCR-LFD) for a rapid diagnosis of melioidosis.

BACKGROUND: Melioidosis is an infectious disease caused by Burkholderia pseudomallei. Septicemic melioidosis patients have a high mortality rate within 48 hours. OBJECTIVE: To develop a polymerase chain reaction (PCR) combined with a lateral flow dipstick (LFD) assay for detection of B. pseudomallei in blood samples. METHODS: The PCR with wcbG gene primers and a PCR-LFD test were developed. The specificity and sensitivity were determined using the B. pseudomallei and other bacterial DNAs. They were evaluated using 43 B. pseudomallei positive blood samples and another 43 blood samples positive for other microbial infections. RESULTS: The detection limit of the PCR-LFD test was 50 fg of bacterial gDNA or 1.0 CFU per 200 µl of blood. All B. pseudomallei were positive while B. thailandensis and selected gram-negative bacterial strains were negative. The PCR-LFD gave all positives with all 43 B. pseudomallei culture positive patient blood samples and all negative with 43 blood samples that were culture positive for K. pneumoniae, E. gallinarum, E. faecium, E. coli, S. aureus, A. baumannii, A. hydrophila, S. haemolyticus, S. pneumoniae, P. aeruginosa, E. cloacae, S. hominis, E. aerogenes, P. mirabilis, C. neoformans, C. albicans, A. caviae, E. faecalis and K. variicola. CONCLUSION: The developed PCR-LFD assay provided 100% sensitivity and 100% specificity compared to the conventional blood culture. The technique took only 1.5 hours that is easy and quick to perform compared to the 3-7 days of culture method. The new method of PCR with LFD could facilitate the detection to be a semi-point-of-care testing (POCT).

Co-evolutionary Signals Identify Burkholderia pseudomallei Survival Strategies in a Hostile Environment.

The soil bacterium Burkholderia pseudomallei is the causative agent of melioidosis and a significant cause of human morbidity and mortality in many tropical and subtropical countries. The species notoriously survives harsh environmental conditions but the genetic architecture for these adaptations remains unclear. Here we employed a powerful combination of genome-wide epistasis and co-selection studies (2,011 genomes), condition-wide transcriptome analyses (82 diverse conditions), and a gene knockout assay to uncover signals of "co-selection"-that is a combination of genetic markers that have been repeatedly selected together through B. pseudomallei evolution. These enabled us to identify 13,061 mutation pairs under co-selection in distinct genes and noncoding RNA. Genes under co-selection displayed marked expression correlation when B. pseudomallei was subjected to physical stress conditions, highlighting the conditions as one of the major evolutionary driving forces for this bacterium. We identified a putative adhesin (BPSL1661) as a hub of co-selection signals, experimentally confirmed a BPSL1661 role under nutrient deprivation, and explored the functional basis of co-selection gene network surrounding BPSL1661 in facilitating the bacterial survival under nutrient depletion. Our findings suggest that nutrient-limited conditions have been the common selection pressure acting on this species, and allelic variation of BPSL1661 may have promoted B. pseudomallei survival during harsh environmental conditions by facilitating bacterial adherence to different surfaces, cells, or living hosts.

Amidine-Based Cationic Conjugated Oligoelectrolytes with Antimicrobial Activity against Pseudomonas aeruginosa Biofilms.

Pseudomonas aeruginosa is an opportunistic Gram-negative bacterium that can cause high-morbidity infections. Due to its robust, flexible genome and ability to form biofilms, it can evade and rapidly develop resistance to antibiotics. Cationic conjugated oligoelectrolytes (COEs) have emerged as a promising class of antimicrobials. Herein, we report a series of amidine-containing COEs with high selectivity for bacteria. From this series, we identified 1b as the most active compound against P. aeruginosa (minimum inhibitory concentration (MIC) = 2 µg/mL) with low cytotoxicity (IC50 (HepG2) = 1024 µg/mL). The activity of 1b was not affected by known drug-resistant phenotypes of 100 diverse P. aeruginosa isolates. Moreover, 1b is bactericidal with a low propensity for P. aeruginosa to develop resistance. Furthermore, 1b is also able to inhibit biofilm formation at subinhibitory concentrations and kills P. aeruginosa in established biofilms. The in vivo efficacy of 1b was demonstrated in biofilm-associated murine wound infection models.

A Review of Omadacycline for Potential Utility in the Military Health System for the Treatment of Wound Infections.

INTRODUCTION: Combat-related wound infections complicate the recovery of wounded military personnel, contributing to overall morbidity and mortality. Wound infections in combat settings present unique challenges because of the size and depth of the wounds, the need to administer emergency care in the field, and the need for subsequent treatment in military facilities. Given the increase in multidrug-resistant pathogens, a novel, broad-spectrum antibiotic is desired across this continuum of care when the standard of care fails. Omadacycline was FDA-approved in 2018 for treatment of adults with acute bacterial skin and skin structure infections (ABSSSI), as well as community-acquired bacterial pneumonia (CABP). It is a broad-spectrum antibiotic with activity against gram-positive, gram-negative, and atypical bacterial pathogens, including multidrug-resistant species. Omadacycline can overcome commonly reported tetracycline resistance mechanisms, ribosomal protection proteins, and efflux pumps, and is available in once-daily intravenous or oral formulations. In this review, we discuss the potential role of omadacycline, which is included in the Department of Defense Formulary, in the context of combat wound infections. MATERIALS AND METHODS: A literature review was undertaken for manuscripts published before July 21, 2023. This included a series of publications found via PubMed and a bibliography made publicly available on the Paratek Pharmaceuticals, Inc. website. Publications presenting primary data published in English on omadacycline in relation to ESKAPEE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, and Enterobacter species) pathogens and Clostridioides difficile, including in vitro, in vivo, and clinical data were included. RESULTS: Of 260 identified records, 66 were included for evidence review. Omadacycline has in vitro activity against almost all the ESKAPEE pathogens, apart from P. aeruginosa. Importantly, it has activity against the four most prevalent bacterial pathogens that cause wound infections in the military healthcare system: S. aureus, including methicillin-resistant S. aureus, A. baumannii, K. pneumoniae, and E. coli. In vivo studies in rats have shown that omadacycline is rapidly distributed in most tissues, with the highest tissue-to-blood concentration ratios in bone mineral. The clinical efficacy of omadacycline has been assessed in three separate Phase 3 studies in patients with ABSSSI (OASIS-1 and OASIS-2) and with CABP (OPTIC). Overall, omadacycline has an established safety profile in the treatment of both ABSSSI and CABP. CONCLUSIONS: Omadacycline has broad-spectrum activity, the option to be orally administered and an established safety profile, making it a potentially attractive replacement for moxifloxacin in the military individual first aid kit, especially when accounting for the increasing resistance to fluoroquinolones. Further studies and clinical evaluation are warranted to support broader use of omadacycline to treat combat wound infections in the military healthcare system.

Transcriptomics Analysis Uncovers Transient Ceftazidime Tolerance in Burkholderia Biofilms.

Burkholderia pseudomallei is an etiological agent of melioidosis, a severe community-acquired infectious disease. B. pseudomallei strain K96243 is sensitive to the drug ceftazidime (CAZ), but has been shown to exhibit transient CAZ tolerance when in a biofilm form. To investigate an observed shift in gene expression profile during CAZ tolerance condition and to better understand the mechanistic aspects of this transient tolerance, RNA-sequencing was performed on B. pseudomallei K96243 from the following three states: planktonic, biofilm, and planktonic shedding. Results indicated that the expression of 651 genes (10.97%) were significantly changed in both biofilm (resistant) and planktonic shedding (sensitive) cells in comparison to the planktonic state. The top four highly expressed genes identified in both states are associated with nitrosative stress response (BPSL2368), Fe-S homeostasis (BPSL2369), and nitrate respiration (BPSS1154 and BPSS1158). Additionally, five orthologous genes, BPSL2370-BPSL2374, implicated in Fe-S cluster biogenesis, and another gene, BPSL2863, involved in DNA-binding of the stress protein ferritin, were shown to increase expression by RT-qPCR. The shift in gene expression was especially prominent at the late stages of biofilm growth (72 and 96 h), specifically in the biofilm-challenged CAZ survivor cells. This suggested that in response to stress in a biofilm, differential expression of these genes may support development of the CAZ tolerance in Burkholderia. The application of iron chelator deferoxamine (DFO) to the biofilm caused a significant reduction in biofilm formation and associated CAZ tolerance. Therefore, the shift in Fe-S metabolism when B. pseudomallei is in a biofilm may help stabilize the levels of reactive oxygen species (ROS), thereby limiting tolerance to CAZ.

Temporal proteomic profiling reveals changes that support Burkholderia biofilms.

Melioidosis associated with opportunistic pathogen Burkholderia pseudomallei imparts a huge medical burden in Southeast Asia and Australia. At present there is no available human vaccine that protects against B. pseudomallei infection and antibiotic treatments are limited particularly for drug-resistant strains and bacteria in biofilm forms. Biofilm forming bacteria exhibit phenotypic features drastically different to their planktonic states, often exhibiting a diminished response to antimicrobial therapies. Our earlier work on global profiling of bacterial biofilms using transcriptomics and proteomics revealed transcript-decoupled protein abundance in bacterial biofilms. Here we employed reverse phase liquid chromatography tandem mass spectrometry (LC-MS/MS) to deduce temporal proteomic differences in planktonic and biofilm forms of Burkholderia thailandensis, which is weakly surrogate model of pathogenic B. pseudomallei as sharing a key element in genomic similarity. The proteomic analysis of B. thailandensis in biofilm versus planktonic states revealed that proteome changes support biofilm survival through decreased abundance of metabolic proteins while increased abundance of stress-related proteins. Interestingly, the protein abundance including for the transcription protein TEX, outer periplasmic TolB protein, and the exopolyphosphatase reveal adaption in bacterial biofilms that facilitate antibiotic tolerance through a non-specific mechanism. The present proteomics study of B. thailandensis biofilms provides a global snapshot of protein abundance differences and antimicrobial sensitivities in planktonic and sessile bacteria.