Growing Burkholderia pseudomallei in Biofilm Stimulating Conditions Significantly Induces Antimicrobial Resistance

BACKGROUND: Burkholderia pseudomallei, a gram-negative bacterium that causes melioidosis, was reported to produce biofilm. As the disease causes high relapse rate when compared to other bacterial infections, it therefore might be due to the reactivation of the biofilm forming bacteria which also provided resistance to antimicrobial agents. However, the mechanism on how biofilm can provide tolerance to antimicrobials is still unclear. METHODOLOGY/PRINCIPAL FINDINGS: The change in resistance of B. pseudomallei to doxycycline, ceftazidime, imipenem, and trimethoprim/sulfamethoxazole during biofilm formation were measured as minimum biofilm elimination concentration (MBEC) in 50 soil and clinical isolates and also in capsule, flagellin, LPS and biofilm mutants. Almost all planktonic isolates were susceptible to all agents studied. In contrast, when they were grown in the condition that induced biofilm formation, they were markedly resistant to all antimicrobial agents even though the amount of biofilm production was not the same. The capsule and O-side chains of LPS mutants had no effect on biofilm formation whereas the flagellin-defective mutant markedly reduced in biofilm production. No alteration of LPS profiles was observed when susceptible form was changed to resistance. The higher amount of N-acyl homoserine lactones (AHLs) was detected in the high biofilm-producing isolates. Interestingly, the biofilm mutant which produced a very low amount of biofilm and was sensitive to antimicrobial agents significantly resisted those agents when grown in biofilm inducing condition. CONCLUSIONS/SIGNIFICANCE: The possible drug resistance mechanism of biofilm mutants and other isolates is not by having biofilm but rather from some factors that up-regulated when biofilm formation genes were stimulated. The understanding of genes related to this situation may lead us to prevent B. pseudomallei biofilms leading to the relapse of melioidosis

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.Peer reviewe

Genomic Islands as a Marker to Differentiate between Clinical and Environmental Burkholderia pseudomallei

Burkholderia pseudomallei, as a saprophytic bacterium that can cause a severe sepsis disease named melioidosis, has preserved several extra genes in its genome for survival. The sequenced genome of the organism showed high diversity contributed mainly from genomic islands (GIs). Comparative genome hybridization (CGH) of 3 clinical and 2 environmental isolates, using whole genome microarrays based on B. pseudomallei K96243 genes, revealed a difference in the presence of genomic islands between clinical and environmental isolates. The largest GI, GI8, of B. pseudomallei was observed as a 2 sub-GI named GIs8.1 and 8.2 with distinguishable %GC content and unequal presence in the genome. GIs8.1, 8.2 and 15 were found to be more common in clinical isolates. A new GI, GI16c, was detected on chromosome 2. Presences of GIs8.1, 8.2, 15 and 16c were evaluated in 70 environmental and 64 clinical isolates using PCR assays. A combination of GIs8.1 and 16c (positivity of either GI) was detected in 70% of clinical isolates and 11.4% of environmental isolates (P<0.001). Using BALB/c mice model, no significant difference of time to mortality was observed between K96243 isolate and three isolates without GIs under evaluation (P>0.05). Some virulence genes located in the absent GIs and the difference of GIs seems to contribute less to bacterial virulence. The PCR detection of 2 GIs could be used as a cost effective and rapid tool to detect potentially virulent isolates that were contaminated in soil

Cloning, expression, and characterization of a peptidoglycan hydrolase from the Burkholderia pseudomallei phage ST79.

The lytic phage ST79 of Burkholderia pseudomallei can lyse a broad range of its host including antibiotic resistant isolates from within using a set of proteins, holin, lysB, lysC and endolysin, a peptidoglycan (PG) hydrolase enzyme. The phage ST79 endolysin gene identified as peptidase M15A was cloned, expressed and purified to evaluate its potential to lyse pathogenic bacteria. The molecular size of the purified enzyme is approximately 18 kDa and the in silico study cited here indicated the presence of a zinc-binding domain predicted to be a member of the subfamily A of a metallopeptidase. Its activity, however, was reduced by the presence of Zn(2+). When Escherichia coli PG was used as a substrate and subjected to digestion for 5 min with 3 μg/ml of enzyme, the peptidase M15A showed 2 times higher in lysis efficiency when compared to the commercial lysozyme. The enzyme works in a broad alkaligenic pH range of 7.5-9.0 and temperatures from 25 to 42 °C. The enzyme was able to lyse 18 Gram-negative bacteria in which the outer membrane was permeabilized by chloroform treatment. Interestingly, it also lysed Enterococcus sp., but not other Gram-positive bacteria. In general, endolysin cannot lyse Gram-negative bacteria from outside, however, the cationic amphipathic C-terminal in some endolysins showed permeability to Gram-negative outer membranes. Genetically engineered ST79 peptidase M15A that showed a broad spectrum against Gram-negative bacterial PG or, in combination with an antibiotic the same way as combined drug methodology, could facilitate an effective treatment of severe or antibiotic resistant cases

Immunostimulatory CpG Oligodeoxynucleotide Confers Protection in a Murine Model of Infection with Burkholderia pseudomallei

Although CpG oligodeoxynucleotides (CpG ODNs) are known to enhance resistance against infection in a number of animal models, little is known about the CpG-induced protection against acute fatal sepsis such as that associated with the highly virulent bacterium Burkholderia pseudomallei. We previously demonstrated in an in vitro study that immunostimulatory CpG ODN 1826 enhances phagocytosis of B. pseudomallei and induces nitric oxide synthase and nitric oxide production by mouse macrophages. In the present study, CpG ODN 1826 given intramuscularly to BALB/c mice 2 to 10 days prior to B. pseudomallei challenge conferred better than 90% protection. CpG ODN 1826 given 2 days before the bacterial challenge rapidly enhanced the innate immunity of these animals, judging from the elevated serum levels of interleukin-12 (IL-12)p70 and gamma interferon (IFN-γ) over the baseline values. No bacteremia was detected on day 2 in 85 to 90% of the CpG-treated animals, whereas more than 80% of the untreated animals exhibited heavy bacterial loads. Although marked elevation of IFN-γ was found consistently in the infected animals 2 days after the bacterial challenge, it was ameliorated by the CpG ODN 1826 pretreatment (P = 0.0002). Taken together, the kinetics of bacteremia and cytokine profiles presented are compatible with the possibility that protection by CpG ODN 1826 against acute fatal septicemic melioidosis in this animal model is associated with a reduction of bacterial load and interference with the potential detrimental effect of the robust production of proinflammatory cytokines associated with B. pseudomallei multiplication

Bacterial Loads and Antibody Responses in BALB/c Mice Infected with Low and High Doses of Burkholderia pseudomallei

Profiles of Burkholderia pseudomallei persistence and antibodies in blood, spleen, liver, and lungs of infected BALB/c mice were investigated. Animals were infected intraperitoneally with low (6 colony-forming units) or high (230 colony-forming units) doses of B. pseudomallei. In the high-dose infected group, bacteria were found by culture in 100% of blood, liver, spleen and lung samples at 24 and 48 hours after infections; blood samples were 100% positive by polymerase chain reaction after 60 hours. Antibody responses in the high-dose infected group were low. These responses were detected in the low-dose infected group after 5 days, peaked at 7–14 days, and showed persistence until 28 days post-infection. Bacterial loads and antibody profiles varied according to the level of bacterial infections. This kinetic study, although in animals, provides crucial knowledge that might be useful for the development of a sensitive and specific diagnostic assay for patients with melioidosis

Cloning, expression, and characterization of a peptidoglycan hydrolase from the Burkholderia pseudomallei phage ST79.

The lytic phage ST79 of Burkholderia pseudomallei can lyse a broad range of its host including antibiotic resistant isolates from within using a set of proteins, holin, lysB, lysC and endolysin, a peptidoglycan (PG) hydrolase enzyme. The phage ST79 endolysin gene identified as peptidase M15A was cloned, expressed and purified to evaluate its potential to lyse pathogenic bacteria. The molecular size of the purified enzyme is approximately 18 kDa and the in silico study cited here indicated the presence of a zinc-binding domain predicted to be a member of the subfamily A of a metallopeptidase. Its activity, however, was reduced by the presence of Zn(2+). When Escherichia coli PG was used as a substrate and subjected to digestion for 5 min with 3 μg/ml of enzyme, the peptidase M15A showed 2 times higher in lysis efficiency when compared to the commercial lysozyme. The enzyme works in a broad alkaligenic pH range of 7.5-9.0 and temperatures from 25 to 42 °C. The enzyme was able to lyse 18 Gram-negative bacteria in which the outer membrane was permeabilized by chloroform treatment. Interestingly, it also lysed Enterococcus sp., but not other Gram-positive bacteria. In general, endolysin cannot lyse Gram-negative bacteria from outside, however, the cationic amphipathic C-terminal in some endolysins showed permeability to Gram-negative outer membranes. Genetically engineered ST79 peptidase M15A that showed a broad spectrum against Gram-negative bacterial PG or, in combination with an antibiotic the same way as combined drug methodology, could facilitate an effective treatment of severe or antibiotic resistant cases