Complete genome sequences of paired isogenic Burkholderia pseudomallei isolated from a Thai pediatric patient with a urinary tract infection.

Burkholderia pseudomallei is the causative agent of melioidosis, the disease endemic in Southeast Asia and northern Australia. We report complete genome sequences of paired isogenic B. pseudomallei isolated from a 12-year-old Thai male presenting with acute urinary tract infection before (SCBP001) and after (SCBP007) a decrease in susceptibility to ceftazidime.

CRISPR Interference-Mediated Silencing of the mmpL3 Gene in Mycobacterium smegmatis and Its Impact on Antimicrobial Susceptibility.

BACKGROUND: The discovery of novel therapeutic agents, especially those targeting mycobacterial membrane protein large 3 (mmpL3), has shown promise. In this study, the CRISPR interference-Streptococcus thermophilus nuclease-deactivated Cas9 (CRISPRi-dCas9Sth1) system was utilized to suppress mmpL3 expression in Mycobacterium smegmatis, and its impacts on susceptibility to antimicrobial agents were evaluated. METHODS: The repression of the mmpL3 gene was confirmed by RT-qPCR. The essentiality, growth curve, viability, and antimicrobial susceptibility of the mmpL3 knockdown strain were investigated. RESULTS: mmpL3 silencing was achieved by utilizing 0.5 and 1 ng/mL anhydrotetracycline (ATc), resulting in reductions in the expression of 60.4% and 74.4%, respectively. mmpL3 silencing led to a significant decrease in bacterial viability when combined with one-half of the minimal inhibitory concentrations (MICs) of rifampicin, rifabutin, ceftriaxone, or isoniazid, along with 0.1 or 0.5 ng/mL ATc (p < 0.05). However, no significant difference was observed for clarithromycin or amikacin. CONCLUSIONS: The downregulation of the mmpL3 gene in mycobacteria was achieved through the use of CRISPRi-dCas9Sth1, resulting in growth deficiencies and resensitization to certain antimicrobial agents. The impact was dependent upon the level of gene expression.

Non-replicative phage particles delivering CRISPR-Cas9 to target major blaCTX-M variants.

Cluster regularly interspaced short palindromic repeats and CRISPR associated protein 9 (CRISPR-Cas9) is a promising tool for antimicrobial re-sensitization by inactivating antimicrobial resistance (AMR) genes of bacteria. Here, we programmed CRISPR-Cas9 with common spacers to target predominant blaCTX-M variants in group 1 and group 9 and their promoter in an Escherichia coli model. The CRISPR-Cas9 was delivered by non-replicative phagemid particles from a two-step process, including insertion of spacer in CRISPR and construction of phagemid vector. Spacers targeting blaCTX-M promoters and internal sequences of blaCTX-M group 1 (blaCTX-M-15 and -55) and group 9 (blaCTX-M-14, -27, -65, and -90) were cloned into pCRISPR and phagemid pRC319 for spacer evaluation and phagemid particle production. Re-sensitization and plasmid clearance were mediated by the spacers targeting internal sequences of each group, resulting in 3 log10 to 4 log10 reduction of the ratio of resistant cells, but not by those targeting the promoters. The CRISPR-Cas9 delivered by modified ΦRC319 particles were capable of re-sensitizing E. coli K-12 carrying either blaCTX-M group 1 or group 9 in a dose-dependent manner from 0.1 to 100 multiplicity of infection (MOI). In conclusion, CRISPR-Cas9 system programmed with well-designed spacers targeting multiple variants of AMR gene along with a phage-based delivery system could eliminate the widespread blaCTX-M genes for efficacy restoration of available third-generation cephalosporins by reversal of resistance in bacteria.

GC-072: A Novel Therapeutic Candidate for Oral Treatment of Melioidosis and Infections Caused by Select Biothreat Pathogens.

Burkholderia pseudomallei (B. pseudomallei), the etiological agent of melioidosis, is a Gram-negative bacterium with additional concern as a biothreat pathogen. The mortality rate from B. pseudomallei varies depending on the type of infection and extent of available health care, but in the case of septicemia left untreated it can range from 50 - 90%. Current therapy for melioidosis is biphasic, consisting of parenteral acute-phase treatment for two weeks or longer, followed by oral eradication-phase treatment lasting several months. An effective oral therapeutic for outpatient treatment of acute-phase melioidosis is needed. GC-072 is a potent, 4-oxoquinolizine antibiotic with selective inhibitory activity against bacterial topoisomerases. GC-072 has demonstrated in vitro potency against susceptible and drug-resistant strains of B. pseudomallei and is also active against Burkholderia mallei, Bacillus anthracis, Yersinia pestis, and Francisella tularensis GC-072 is bactericidal both extra- and intracellularly, with rapid killing noted within a few hours and reduced development of resistance compared to ceftazidime. GC-072, delivered intragastrically to mimic oral administration, promoted dose-dependent survival in mice using lethal inhalational models of B. pseudomallei infection following exposure to a 24 or 339 LD50 challenge with B. pseudomallei strain 1026b. Overall, GC-072 appears to be a strong candidate for first-line, oral treatment of melioidosis.

Burkholderia pseudomallei acquired ceftazidime resistance due to gene duplication and amplification.

Ceftazidime (CAZ) is the antibiotic of choice for the treatment of Burkholderia pseudomallei infection (melioidosis). The chromosomally-encoded PenA ß-lactamase possesses weak cephalosporinase activity. The wild-type penA gene confers clinically significant CAZ resistance only when overexpressed due to a promoter mutation, transcriptional antitermination or by gene duplication and amplification (GDA). Here we characterise a reversible 33-kb GDA event involving wild-type penA in a CAZ-resistant B. pseudomallei clinical isolate from Thailand. We show that duplication arises from exchanges between short (<10 bp) chromosomal sequences, which in this example consist of 4-bp repeats flanked by 3-bp inverted repeats. GDA involving ß-lactamases may be a common CAZ resistance mechanism in B. pseudomallei.

Transcriptional and post-transcriptional regulation of PenA ß-lactamase in acquired Burkholderia pseudomallei ß-lactam resistance.

Therapy of Burkholderia pseudomallei acute infections is largely limited to a few ß-lactam antibiotics such as ceftazidime or meropenem. Although relatively rare, resistance emergence during therapy leads to treatment failures with high mortality rates. In the absence of acquired external resistance determinants in B. pseudomallei emergence of ß-lactam resistance is invariably caused by mutational modification of genomically encoded factors. These include the deletion of the ceftazidime target penicillin-binding protein 3 or amino acid changes in the Class A PenA ß-lactamase that expand its substrate spectrum, as well as penA gene duplication and amplification or its overexpression via transcriptional up-regulation. Evidence is presented that penA is co-transcribed with the upstream nlpD1 gene, that the transcriptional terminator for nlpD1 serves as a penA attenuator and that generation of a new promoter immediately upstream of the terminator/attenuator by a conserved G to A transition leads to anti-termination and thus constitutive PenA expression and extended ß-lactam resistance. Further evidence obtained with the extensively ß-lactam resistant clinical isolate Bp1651 shows that in addition to PenA overexpression and structural mutations other adaptive mechanisms contribute to intrinsic and acquired B. pseudomallei ß-lactam resistance.

Molecular determinants of Burkholderia pseudomallei BpeEF-OprC efflux pump expression.

Burkholderia pseudomallei, the cause of melioidosis, is intrinsically resistant to many antibiotics. Acquired multidrug resistance, including resistance to doxycycline and co-trimoxazole used for melioidosis eradication phase therapy, is mainly attributed to constitutive expression of the BpeEF-OprC efflux pump. Constitutive expression of this pump is caused by mutations affecting two highly similar LysR-type transcriptional regulators (LTTR), BpeT and BpeS, but their interaction with the regulatory region governing BpeEF-OprC expression has not yet been studied. The bpeE-bpeF-oprC genes are distally located in the llpE-bpeE-bpeF-oprC operon. The llpE gene encodes a putative lipase/esterase of unknown function. We show that in a bpeT mutant llpE is constitutively co-transcribed with bpeE-bpeF-oprC. As expected from previous studies with B. cenocepacia, deletion of llpE does not affect antibiotic efflux. Using transcriptional bpeE'-lacZ fusions, we demonstrate that the 188 bp bpeT-llpE intergenic region located between bpeT and the llpE-bpeE-bpeF-oprC operon contains regulatory elements needed for control of bpeT and llpE-bpeE-bpeF-oprC operon expression. By native polyacrylamide gel electrophoresis and electrophoretic mobility shift assays with purified recombinant BpeT and BpeS proteins, we show BpeT and BpeS form oligomers that share a 14 bp binding site overlapping the essential region required for llpE-bpeE-bpeF-oprC expression. The binding site contains the conserved T-N11-A LTTR box motif involved in binding of LysR proteins, which in concert with two other possible LTTR boxes may mediate BpeT and BpeS regulation of BpeEF-OprC expression. These studies form the basis for further investigation of BpeEF-OprC expression and regulation at the molecular level by yet unknown external stimuli.

Outer Membrane Vesicle Vaccines from Biosafe Surrogates Prevent Acute Lethal Glanders in Mice.

Burkholderia mallei is a host-adapted Gram-negative mammalian pathogen that causes the severe disease glanders. Glanders can manifest as a rapid acute progression or a chronic debilitating syndrome primarily affecting solipeds and humans in close association with infected animals. In USA, B. mallei is classified as one of the most important bacterial biothreat agents. Presently, there is no licensed glanders vaccine available for humans or animals. In this work, outer membrane vesicles (OMVs) were isolated from three attenuated biosafe bacterial strains, Burkholderia pseudomallei Bp82, B. thailandensis E555, and B. thailandensis TxDOH and used to vaccinate mice. B. thailandensis OMVs induced significantly higher antibody responses that were investigated. B. mallei specific serum antibody responses were of higher magnitude in mice vaccinated with B. thailandensis OMVs compared to levels in mice vaccinated with B. pseudomallei OMVs. OMVs derived from biosafe strains protected mice from acute lethal glanders with vesicles from the two B. thailandensis strains affording significant protection (>90%) up to 35 days post-infection with some up to 60 days. Organ loads from 35-day survivors indicated bacteria colonization of the lungs, liver, and spleen while those from 60 days had high CFUs in the spleens. The highest antibody producing vaccine (B. thailandensis E555 OMVs) also protected C57BL/6 mice from acute inhalational glanders with evidence of full protection.

Mechanisms of Resistance to Folate Pathway Inhibitors in Burkholderia pseudomallei: Deviation from the Norm.

The trimethoprim and sulfamethoxazole combination, co-trimoxazole, plays a vital role in the treatment of Burkholderia pseudomallei infections. Previous studies demonstrated that the B. pseudomallei BpeEF-OprC efflux pump confers widespread trimethoprim resistance in clinical and environmental isolates, but this is not accompanied by significant resistance to co-trimoxazole. Using the excluded select-agent strain B. pseudomallei Bp82, we now show that in vitro acquired trimethoprim versus co-trimoxazole resistance is mainly mediated by constitutive BpeEF-OprC expression due to bpeT mutations or by BpeEF-OprC overexpression due to bpeS mutations. Mutations in bpeT affect the carboxy-terminal effector-binding domain of the BpeT LysR-type activator protein. Trimethoprim resistance can also be mediated by dihydrofolate reductase (FolA) target mutations, but this occurs rarely unless BpeEF-OprC is absent. BpeS is a transcriptional regulator that is 62% identical to BpeT. Mutations affecting the BpeS DNA-binding or carboxy-terminal effector-binding domains result in constitutive BpeEF-OprC overexpression, leading to trimethoprim and sulfamethoxazole efflux and thus to co-trimoxazole resistance. The majority of laboratory-selected co-trimoxazole-resistant mutants often also contain mutations in folM, encoding a pterin reductase. Genetic analyses of these mutants established that both bpeS mutations and folM mutations contribute to co-trimoxazole resistance, although the exact role of folM remains to be determined. Mutations affecting bpeT, bpeS, and folM are common in co-trimoxazole-resistant clinical isolates, indicating that mutations affecting these genes are clinically significant. Co-trimoxazole resistance in B. pseudomallei is a complex phenomenon, which may explain why resistance to this drug is rare in this bacterium.IMPORTANCEBurkholderia pseudomallei causes melioidosis, a tropical disease that is difficult to treat. The bacterium's resistance to antibiotics limits therapeutic options. The paucity of orally available drugs further complicates therapy. The oral drug of choice is co-trimoxazole, a combination of trimethoprim and sulfamethoxazole. These antibiotics target two distinct enzymes, FolA (dihydrofolate reductase) and FolP (dihydropteroate synthase), in the bacterial tetrahydrofolate biosynthetic pathway. Although co-trimoxazole resistance is minimized due to two-target inhibition, bacterial resistance due to folA and folP mutations does occur. Co-trimoxazole resistance in B. pseudomallei is rare and has not yet been studied. Co-trimoxazole resistance in this bacterium employs a novel strategy involving differential regulation of BpeEF-OprC efflux pump expression that determines the drug resistance profile. Contributing are mutations affecting folA, but not folP, and folM, a folate pathway-associated gene whose function is not yet well understood and which has not been previously implicated in folate inhibitor resistance in clinical isolates.

Antibiotic Resistance Markers in Burkholderia pseudomallei Strain Bp1651 Identified by Genome Sequence Analysis.

Burkholderia pseudomallei Bp1651 is resistant to several classes of antibiotics that are usually effective for treatment of melioidosis, including tetracyclines, sulfonamides, and ß-lactams such as penicillins (amoxicillin-clavulanic acid), cephalosporins (ceftazidime), and carbapenems (imipenem and meropenem). We sequenced, assembled, and annotated the Bp1651 genome and analyzed the sequence using comparative genomic analyses with susceptible strains, keyword searches of the annotation, publicly available antimicrobial resistance prediction tools, and published reports. More than 100 genes in the Bp1651 sequence were identified as potentially contributing to antimicrobial resistance. Most notably, we identified three previously uncharacterized point mutations in penA, which codes for a class A ß-lactamase and was previously implicated in resistance to ß-lactam antibiotics. The mutations result in amino acid changes T147A, D240G, and V261I. When individually introduced into select agent-excluded B. pseudomallei strain Bp82, D240G was found to contribute to ceftazidime resistance and T147A contributed to amoxicillin-clavulanic acid and imipenem resistance. This study provides the first evidence that mutations in penA may alter susceptibility to carbapenems in B. pseudomallei Another mutation of interest was a point mutation affecting the dihydrofolate reductase gene folA, which likely explains the trimethoprim resistance of this strain. Bp1651 was susceptible to aminoglycosides likely because of a frameshift in the amrB gene, the transporter subunit of the AmrAB-OprA efflux pump. These findings expand the role of penA to include resistance to carbapenems and may assist in the development of molecular diagnostics that predict antimicrobial resistance and provide guidance for treatment of melioidosis.

Characterization of BPSS1521 (bprD), a regulator of Burkholderia pseudomallei virulence gene expression in the mouse model.

The Gram-negative saprophytic bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a severe infectious disease of both humans and animals. Severity of the disease is thought to be dependent on both the health status of the host, including diabetes mellitus and kidney disease, and bacterial-derived factors. To identify the bacterial factors important during an acute infection, gene expression profiles in the spleen, lung, and liver of BALB/c (Th2 prototype) and C57BL/6 mice (Th1 prototype) were determined using DNA microarrays. This analysis identified BPSS1521 (bprD), a predicted transcriptional regulator located in the type III secretion system (T3SS-3) operon, to be up regulated, specifically in C57BL/6 mice. BALB/c mice infected with a bprD mutant showed a shorter time to death and increased inflammation, as determined by histopathological analysis and enumeration of bacteria in the spleen. Elevated numbers of multinucleated giant cells (MNGCs), which is the hallmark of melioidosis, were detected in both the wild-type and the bprD mutants; a similar elevation occurs in melioidosis patients. One striking observation was the increased expression of BPSS1520 (bprC), located downstream of bprD, in the bprD mutant. BprC is a regulator of T6SS-1 that is required for the virulence of B. pseudomallei in murine infection models. Deletion of bprD led to the overexpression of bprC and a decreased time to death. bprD expression was elevated in C57BL/6--as compared to BALB/c--mice, suggesting a role for BprD in the natural resistance of C57BL/6 mice to B. pseudomallei. Ultimately, this analysis using mice with different immune backgrounds may enhance our understanding of the outcomes of infection in a variety of models.

Effective screening for double heterozygosity of Hb E/alpha0-thalassemia.

One hundred and forty-one blood samples of hemoglobin E (Hb E) carriers were collected to define suitable cutoff values of Hb E level, mean corpuscular volume (MCV), and mean corpuscular hemoglobin (MCH) as screening indicators for detecting individuals with double heterozygosity for Hb E/alpha(o)-thalassemia. Based on the values that gave 100% sensitivity, Hb E < 26%, MCV < 74 fl, and MCH < 24 pg were selected. Further validation of these selected values in additional 152 heterozygous Hb E pregnant women revealed 100% sensitivity, 86.2% specificity, and a 25.9% positive predictive value (PPV) for using Hb E cutoff point only, meanwhile, 100% sensitivity, 83.4% specificity, and 22.6% PPV were achieved for the MCV cutoff point. In addition, 100% sensitivity, 86.3% specificity, and 25.9% PPV were gained for the MCH cutoff point. Combining Hb E level with either MCV or MCH cutoff points, the specificity and PPV were increased to 95.1% and 50.0%, respectively. It is concluded that Hb E level < 26%, MCV < 74 fl, and MCH < 24 pg could be used for screening alpha(o)-thalassemia in heterozygous Hb E. However, to improve specificity and PPV of the tests, a combination of Hb E level < 26% with either MCV < 74 fl or MCH < 24 pg is recommended.