Characterization of ceftazidime resistance mechanisms in clinical isolates of Burkholderia pseudomallei from Australia

Burkholderia pseudomallei is a gram-negative bacterium that causes the serious human disease, melioidosis. There is no vaccine against melioidosis and it can be fatal if not treated with a specific antibiotic regimen, which typically includes the third-generation cephalosporin, ceftazidime (CAZ). There have been several resistance mechanisms described for B. pseudomallei, of which the best described are amino acid changes that alter substrate specificity in the highly conserved class A β-lactamase, PenA. In the current study, we sequenced penA from isolates sequentially derived from two melioidosis patients with wild-type (1.5 μg/mL) and, subsequently, resistant (16 or ≥256 μg/mL) CAZ phenotypes. We identified two single-nucleotide polymorphisms (SNPs) that directly increased CAZ hydrolysis. One SNP caused an amino acid substitution (C69Y) near the active site of PenA, whereas a second novel SNP was found within the penA promoter region. In both instances, the CAZ resistance phenotype corresponded directly with the SNP genotype. Interestingly, these SNPs appeared after infection and under selection from CAZ chemotherapy. Through heterologous cloning and expression, and subsequent allelic exchange in the native bacterium, we confirmed the role of penA in generating both low-level and high-level CAZ resistance in these clinical isolates. Similar to previous studies, the amino acid substitution altered substrate specificity to other β-lactams, suggesting a potential fitness cost associated with this mutation, a finding that could be exploited to improve therapeutic outcomes in patients harboring CAZ resistant B. pseudomallei. Our study is the first to functionally characterize CAZ resistance in clinical isolates of B. pseudomallei and to provide proven and clinically relevant signatures for monitoring the development of antibiotic resistance in this important pathogen.     

Genomic diversity of Burkholderia pseudomallei clinical isolates: subtractive hybridization reveals a Burkholderia mallei-specific prophage in B. pseudomallei 1026b

Burkholderia pseudomallei is the etiologic agent of the disease melioidosis and is a category B biological threat agent. The genomic sequence of B. pseudomallei K96243 was recently determined, but little is known about the overall genetic diversity of this species. Suppression subtractive hybridization was employed to assess the genetic variability between two distinct clinical isolates of B. pseudomallei, 1026b and K96243. Numerous mobile genetic elements, including a temperate bacteriophage designated phi1026b, were identified among the 1026b-specific suppression subtractive hybridization products. Bacteriophage phi1026b was spontaneously produced by 1026b, and it had a restricted host range, infecting only Burkholderia mallei. It possessed a noncontractile tail, an isometric head, and a linear 54,865-bp genome. The mosaic nature of the phi1026b genome was revealed by comparison with bacteriophage phiE125, a B. mallei-specific bacteriophage produced by Burkholderia thailandensis. The phi1026b genes for DNA packaging, tail morphogenesis, host lysis, integration, and DNA replication were nearly identical to the corresponding genes in phiE125. On the other hand, phi1026b genes involved in head morphogenesis were similar to head morphogenesis genes encoded by Pseudomonas putida and Pseudomonas aeruginosa bacteriophages. Consistent with this observation, immunogold electron microscopy demonstrated that polyclonal antiserum against phiE125 reacted with the tail of phi1026b but not with the head. The results presented here suggest that B. pseudomallei strains are genetically heterogeneous and that bacteriophages are major contributors to the genomic diversity of this species. The bacteriophage characterized in this study may be a useful diagnostic tool for differentiating B. pseudomallei and B. mallei, two closely related biological threat agents.     

Biogeography and genetic diversity of clinical isolates of Burkholderia pseudomallei in Sri Lanka

BackgroundMelioidosis is a potentially fatal infectious disease caused by Burkholderia pseudomallei and the disease is endemic in Southeast Asia and Northern Australia. It has been confirmed as endemic in Sri Lanka. Genomic epidemiology of B. pseudomallei in Sri Lanka is largely unexplored. This study aims to determine the biogeography and genetic diversity of clinical isolates of B. pseudomallei and the phylogenetic and evolutionary relationship of Sri Lankan sequence types (STs) to those found in other endemic regions of Southeast Asia and Oceania.MethodsThe distribution of variably present genetic markers [Burkholderia intracellular motility A (bimA) gene variants bimA_BP/bimA_BM, filamentous hemagglutinin 3 (fhaB3), Yersinia-like fimbrial (YLF) and B. thailandensis-like flagellum and chemotaxis (BTFC) gene clusters and lipopolysaccharide O-antigen type A (LPS type A)] was examined among 310 strains. Multilocus sequence typing (MLST) was done for 84 clinical isolates. The phylogenetic and evolutionary relationship of Sri Lankan STs within Sri Lanka and in relation to those found in other endemic regions of Southeast Asia and Oceania were studied using e BURST, PHYLOViZ and minimum evolutionary analysis.ResultsThe Sri Lankan B. pseudomallei population contained a large proportion of the rare BTFC clade (14.5%) and bimA_BM allele variant (18.5%) with differential geographic distribution. Genotypes fhaB3 and LPSA were found in 80% and 86% respectively. This study reported 43 STs (including 22 novel). e-BURST analysis which include all Sri Lankan STs (71) resulted in four groups, with a large clonal group (group 1) having 46 STs, and 17 singletons. ST1137 was the commonest ST. Several STs were shared with India, Bangladesh and Cambodia.ConclusionThis study demonstrates the usefulness of high-resolution molecular typing to locate isolates within the broad geographical boundaries of B. pseudomallei at a global level and reveals that Sri Lankan isolates are intermediate between Southeast Asia and Oceania.     

Multi locus sequence typing: an informative approach to molecular ecology

The use of multi locus sequence typing in the investigation of Legionella pneumophila isolated in Russia allowed us to obtain new data for molecular-ecological analysis of the current situation. The strains from the groundwater of different distant regions of the country displayed similar allelic profiles. Mutation processes in cooling towers and in the areas of water stagnation in the autonomous water supply systems have a similar direction. The recombination mechanism was predominant in the process of clonal complex formation. An epidemiological assessment of the investigated strains on the basis of comparison of their allelic profiles with the EWGLI database is presented.     

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

BackgroundMelioidosis 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 findingsWe 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.ConclusionThe 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.     

类鼻疽伯克霍尔德致死因子

类鼻疽伯克霍尔德菌是一种胞内感染的革兰阴性杆菌,所导致的疾病称为类鼻疽。迄今为止,还没有针对类鼻疽的疫苗。其致死因子1(BLF1)作为类鼻疽伯克霍尔德菌的重要致病因子,能抑制宿主细胞翻译起始因子e IF4A的解旋酶活性,从而抑制蛋白质合成。BLF1作为e IF4A的抑制剂,有望成为靶向抗肿瘤药物。同时,BLF1具有很强的免疫原性,对其进行结构改造后,可成为类鼻疽疫苗的候选抗原。     

Molecular typing of the Burkholderia pseudomallei strains with different resistance to antibiotics

Wild type strains of Burkholderia pseudomallei, spontaneous mutants with high resistance to fluoroquinolones and ceftazidime, and Tn5-induced mutants with reduced resistance level were studied using polymorphic and gene-specific DNA fingerprinting. Cluster analysis of genomic DNA patterns obtained using PCR with arbitrary primer (5'-GTTTCGCTCC-3') and primer specific to the class I integrase intll gene (5'-CCTCCCGCACGATGATC-3') was performed. According to the DNA pattern conformity, the distinct groups submitted by high-level resistant B. pseudomallei derivatives were revealed by both typing approaches. The obtained results may be useful in searching for molecular markers associated with different types of antimicrobial resistance among pathogenic and related burkholderiae.     

Fine-scale genetic diversity among Burkholderia pseudomallei soil isolates in northeast Thailand

Burkholderia pseudomallei soil isolates from northeast Thailand were genotyped using multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) and multilocus sequence typing (MLST). MLVA identified 19 genotypes within three clades, while MLST revealed two genotypes. These close genetic relationships imply a recent colonization followed by localized expansion, similar to what occurs in an outbreak situation.     

The wbiA locus is required for the 2-O-acetylation of lipopolysaccharides expressed by Burkholderia pseudomallei and Burkholderia thailandensis

Burkholderia pseudomallei and Burkholderia thailandensis express similar O-antigens (O-PS II) in which their 6-deoxy-alpha-L-talopyranosyl (L-6dTalp) residues are variably substituted with O-acetyl groups at the O-2 or O-4 positions. In previous studies we demonstrated that the protective monoclonal antibody, Pp-PS-W, reacted with O-PS II expressed by wild-type B. pseudomallei strains but not by a B. pseudomallei wbiA null mutant. In the present study we demonstrate that WbiA activity is required for the acetylation of the L-6dTalp residues at the O-2 position and that structural modification of O-PS II molecules at this site is critical for recognition by Pp-PS-W.     

In silico analysis of Burkholderia pseudomallei genome sequence for potential drug targets

Recent advances in DNA sequencing technology have enabled elucidation of whole genome information from a plethora of organisms. In parallel with this technology, various bioinformatics tools have driven the comparative analysis of the genome sequences between species and within isolates. While drawing meaningful conclusions from a large amount of raw material, computer-aided identification of suitable targets for further experimental analysis and characterization, has also led to the prediction of non-human homologous essential genes in bacteria as promising candidates for novel drug discovery. Here, we present a comparative genomic analysis to identify essential genes in Burkholderia pseudomallei. Our in silico prediction has identified 312 essential genes which could also be potential drug candidates. These genes encode essential proteins to support the survival of B. pseudomallei including outer-inner membrane and surface structures, regulators, proteins involved in pathogenenicity, adaptation, chaperones as well as degradation of small and macromolecules, energy metabolism, information transfer, central/intermediate/miscellaneous metabolism pathways and some conserved hypothetical proteins of unknown function. Therefore, our in silico approach has enabled rapid screening and identification of potential drug targets for further characterization in the laboratory.     

Outer membrane proteome of Burkholderia pseudomallei and Burkholderia mallei from diverse growth conditions

Burkholderia mallei and Burkholderia pseudomallei are closely related, aerosol-infective human pathogens that cause life-threatening diseases. Biochemical analyses requiring large-scale growth and manipulation at biosafety level 3 under select agent regulations are cumbersome and hazardous. We developed a simple, safe, and rapid method to prepare highly purified outer membrane (OM) fragments from these pathogens. Shotgun proteomic analyses of OMs by trypsin shaving and mass spectrometry identified >155 proteins, the majority of which are clearly outer membrane proteins (OMPs). These included: 13 porins, 4 secretins for virulence factor export, 11 efflux pumps, multiple components of a Type VI secreton, metal transport receptors, polysaccharide exporters, and hypothetical OMPs of unknown function. We also identified 20 OMPs in each pathogen that are abundant under a wide variety of conditions, including in serum and with macrophages, suggesting these are fundamental for growth and survival and may represent prime drug or vaccine targets. Comparison of the OM proteomes of B. mallei and B. pseudomallei showed many similarities but also revealed a few differences, perhaps reflecting evolution of B. mallei away from environmental survival toward host-adaptation.     

Molecular characterization of clinical isolates of Aeromonas species from Malaysia

Background

Aeromonas species are common inhabitants of aquatic environments giving rise to infections in both fish and humans. Identification of aeromonads to the species level is problematic and complex due to their phenotypic and genotypic heterogeneity.

Methodology/Principal Findings

Aeromonas hydrophila or Aeromonas sp were genetically re-identified using a combination of previously published methods targeting GCAT, 16S rDNA and rpoD genes. Characterization based on the genus specific GCAT-PCR showed that 94 (96%) of the 98 strains belonged to the genus Aeromonas. Considering the patterns obtained for the 94 isolates with the 16S rDNA-RFLP identification method, 3 clusters were recognised, i.e. A. caviae (61%), A. hydrophila (17%) and an unknown group (22%) with atypical RFLP restriction patterns. However, the phylogenetic tree constructed with the obtained rpoD sequences showed that 47 strains (50%) clustered with the sequence of the type strain of A. aquariorum, 18 (19%) with A. caviae, 16 (17%) with A. hydrophila, 12 (13%) with A. veronii and one strain (1%) with the type strain of A. trota. PCR investigation revealed the presence of 10 virulence genes in the 94 isolates as: lip (91%), exu (87%), ela (86%), alt (79%), ser (77%), fla (74%), aer (72%), act (43%), aexT (24%) and ast (23%).

Conclusions/Significance

This study emphasizes the importance of using more than one method for the correct identification of Aeromonas strains. The sequences of the rpoD gene enabled the unambiguous identication of the 94 Aeromonas isolates in accordance with results of other recent studies. Aeromonas aquariorum showed to be the most prevalent species (50%) containing an important subset of virulence genes lip/alt/ser/fla/aer. Different combinations of the virulence genes present in the isolates indicate their probable role in the pathogenesis of Aeromonas infections.     

Novel lytic bacteriophages from soil that lyse Burkholderia pseudomallei

Burkholderia pseudomallei is a Gram-negative saprophytic bacterium that causes severe sepsis with a high mortality rate in humans and a vaccine is not available. Bacteriophages are viruses of bacteria that are ubiquitous in nature. Several lysogenic phages of Burkholderia spp. have been found but information is scarce for lytic phages. Six phages, ST2, ST7, ST70, ST79, ST88 and ST96, which lyse B. pseudomallei, were isolated from soil in an endemic area. The phages belong to the Myoviridae family. The range of estimated genome sizes is 24.0-54.6 kb. Phages ST79 and ST96 lysed 71% and 67% of tested B. pseudomallei isolates and formed plaques on Burkholderia mallei but not other tested bacteria, with the exception of closely related Burkholderia thailandensis which was lysed by ST2 and ST96 only. ST79 and ST96 were observed to clear a mid-log culture by lysis within 6 h when infected at a multiplicity of infection of 0.1. As ST79 and ST96 phages effectively lysed B. pseudomallei, their potential use as a biocontrol of B. pseudomallei in the environment or alternative treatment in infected hosts could lead to benefits from phages that are available in nature.     

In silico analyses of penicillin binding proteins in Burkholderia pseudomallei uncovers SNPs with utility for phylogeography,species differentiation,and sequence typing

Burkholderia pseudomallei causes melioidosis. Sequence typing this pathogen can reveal geographical origin and uncover epidemiological associations. Here, we describe B. pseudomallei genes encoding putative penicillin binding proteins (PBPs) and investigate their utility for determining phylogeography and differentiating closely related species. We performed in silico analysis to characterize 10 PBP homologs in B. pseudomallei 1026b. As PBP active site mutations can confer β-lactam resistance in Gram-negative bacteria, PBP sequences in two resistant B. pseudomallei strains were examined for similar alterations. Sequence alignments revealed single amino acid polymorphisms (SAAPs) unique to the multidrug resistant strain Bp1651 in the transpeptidase domains of two PBPs, but not directly within the active sites. Using BLASTn analyses of complete assembled genomes in the NCBI database, we determined genes encoding PBPs were conserved among B. pseudomallei (n = 101) and Burkholderia mallei (n = 26) strains. Within these genes, single nucleotide polymorphisms (SNPs) useful for predicting geographic origin of B. pseudomallei were uncovered. SNPs unique to B. mallei were also identified. Based on 11 SNPs identified in two genes encoding predicted PBP-3s, a dual-locus sequence typing (DLST) scheme was developed. The robustness of this typing scheme was assessed using 1,523 RefSeq genomes from B. pseudomallei (n = 1,442) and B. mallei (n = 81) strains, resulting in 32 sequence types (STs). Compared to multi-locus sequence typing (MLST), the DLST scheme demonstrated less resolution to support the continental separation of Australian B. pseudomallei strains. However, several STs were unique to strains originating from a specific country or region. The phylogeography of Western Hemisphere B. pseudomallei strains was more highly resolved by DLST compared to internal transcribed spacer (ITS) typing, and all B. mallei strains formed a single ST. Conserved genes encoding PBPs in B. pseudomallei are useful for strain typing, can enhance predictions of geographic origin, and differentiate strains of closely related Burkholderia species.     

Investigating Burkholderia cepacia complex populations recovered from Italian maize rhizosphere by multilocus sequence typing

The Burkholderia cepacia complex (BCC) comprises at least nine closely related species of abundant environmental microorganisms. Some of these species are highly spread in the rhizosphere of several crop plants, particularly of maize; additionally, as opportunistic pathogens, strains of the BCC are capable of colonizing humans. We have developed and validated a multilocus sequence typing (MLST) scheme for the BCC. Although widely applied to understand the epidemiology of bacterial pathogens, MLST has seen limited application to the population analysis of species residing in the natural environment; we describe its novel application to BCC populations within maize rhizospheres. 115 BCC isolates were recovered from the roots of different maize cultivars from three different Italian regions over a 9-year period (1994-2002). A total of 44 sequence types (STs) were found of which 41 were novel when compared with existing MLST data which encompassed a global database of 1000 clinical and environmental strains representing nearly 400 STs. In this study of rhizosphere isolates approximately 2.5 isolates per ST was found, comparable to that found for the whole BCC population. Multilocus sequence typing also resolved inaccuracies associated with previous identification of the maize isolates based on recA gene restriction fragment length polymorphims and species-specific polymerase chain reaction. The 115 maize isolates comprised the following BCC species groups, B. ambifaria (39%), BCC6 (29%), BCC5 (10%), B. pyrrocinia (8%), B. cenocepacia IIIB (7%) and B. cepacia (6%), with BCC5 and BCC6 potentially constituting novel species groups within the complex. Closely related clonal complexes of strains were identified within B. cepacia, B. cenocepacia IIIB, BCC5 and BCC6, with one of the BCC5 clonal complexes being distributed across all three sampling sites. Overall, our analysis demonstrates that the maize rhizosphere harbours a massive diversity of novel BCC STs, so that their addition to our global MLST database increased the ST diversity by 10%.     

Membrane-active mechanism of LFchimera against Burkholderia pseudomallei and Burkholderia thailandensis

LFchimera, a construct combining two antimicrobial domains of bovine lactoferrin, lactoferrampin265–284 and lactoferricin17–30, possesses strong bactericidal activity. As yet, no experimental evidence was presented to evaluate the mechanisms of LFchimera against Burkholderia isolates. In this study we analyzed the killing activity of LFchimera on the category B pathogen Burkholderia pseudomallei in comparison to the lesser virulent Burkholderia thailandensis often used as a model for the highly virulent B. pseudomallei. Killing kinetics showed that B. thailandensis E264 was more susceptible for LFchimera than B. pseudomallei 1026b. Interestingly the bactericidal activity of LFchimera appeared highly pH dependent; B. thailandensis killing was completely abolished at and below pH 6.4. FITC-labeled LFchimera caused a rapid accumulation within 15 min in the cytoplasm of both bacterial species. Moreover, freeze-fracture electron microscopy demonstrated extreme effects on the membrane morphology of both bacterial species within 1 h of incubation, accompanied by altered membrane permeability monitored as leakage of nucleotides. These data indicate that the mechanism of action of LFchimera is similar for both species and encompasses disruption of the plasma membrane and subsequently leakage of intracellular nucleotides leading to cell dead.     

Immunobiological properties of Burkholderia pseudomallei and Burkholderia mallei capsular substances

The biopolymer composition, immunotropic and immunogenic properties of the fractions of B. pseudomallei and B. mallei were under study. The first two capsular fractions of these agents were found to be similar in their biopolymer composition that was indicative of their close relations. At the same time the causative agents of glanders proved to have decreased content of high molecular glycoproteids and LPS fragments. In the causative agents of melioidosis, capsular fractions K3 and K4 were characterized by the domination of proteins with a molecular weight of 42-25 kD. Fraction K4 in B. pseudomallei and fraction K1 in B. mallei had pronounced immunosuppressing properties ensuring the protection of encapsulated microbial cells in the body. The biopolymers forming fractions K1, K2, K3 in B. pseudomallei and fraction K2 in B. mallei were characterized by immunomodulating properties.