Single gene target bacterial identification. groEL gene sequencing for discriminating clinical isolates of Burkholderia pseudomallei and Burkholderia thailandensis

Proper identification of Burkholderia pseudomallei and Burkholderia thailandensis is crucial in guiding clinical management of patients with suspected melioidosis, as more than 99% of cases of melioidosis are caused by B. pseudomallei, whereas B. thailandensis is only responsible for causing less than 1% of the cases. However, the difference between the 16S ribosomal RNA gene sequences of B. pseudomallei and that of B. thailandensis is only 1%, and is therefore not discriminative enough for distinguishing the 2 species confidently. In this study, we amplified and sequenced the groEL genes of 7 strains of B. thailandensis and 6 strains of B. pseudomallei, and compared the sequences with 7 other groEL gene sequences of Burkholderia species. BLAST analysis revealed that the putative protein encoded by the groEL gene of B. thailandensis has 99.6%, 99.5%, 98.4%, 98.5%, and 96.5% amino acid identity with the groEL of B. pseudomallei, B. mallei, B. cepacia, B. vietnamiensis, and B. fungorum respectively. The amino acid sequences of GroEL of the strains of B. thailandensis and B. pseudomallei all showed >99.5% amino acid identity with each other. The nucleotide sequence of the groEL gene of any of the strains of B. thailandensis showed >99.8% nucleotide identity with that of any of the other strains of B. thailandensis, and the nucleotide sequence of the groEL gene of any of the strains of B. pseudomallei showed >99.5% nucleotide identity with that of any of the other strains of B. pseudomallei. However, the nucleotide sequence of any of the strains of B. thailandensis showed <97.6% nucleotide identity with any of the strains of B. pseudomallei. The amino acid sequences of GroEL of the 20 strains of Burkholderia species all showed >96% amino acid identity with each other. Furthermore, the nucleotide sequence of the groEL genes of the 2 strains of B. cepacia showed >99.5% nucleotide identity with each other, and the nucleotide sequence of the groEL gene of B. mallei showed >99.5% nucleotide identity with any of the strains of B. pseudomallei. The groEL gene sequence is therefore good for distinguishing between B. thailandensis and B. pseudomallei, and the GroEL amino acid and groEL nucleotide sequences of this single gene locus may potentially be useful for a 2-tier hierarchical identification of medically important Burkholderia at the genus and species levels respectively.     

In Vitro Susceptibilities of Burkholderia mallei in Comparison to Those of Other Pathogenic Burkholderia spp.

The in vitro antimicrobial susceptibilities of isolates of Burkholderia mallei to 16 antibiotics were assessed and compared with the susceptibilities of Burkholderia pseudomallei and Burkholderia cepacia. The antibiotic susceptibility profile of B. mallei resembled that of B. pseudomallei more closely than that of B. cepacia, which corresponds to their similarities in terms of biochemistry, antigenicity, and pathogenicity. Ceftazidime, imipenem, doxycycline, and ciprofloxacin were active against both B. mallei and B. pseudomallei. Gentamicin was active against B. mallei but not against B. pseudomallei. Antibiotics clinically proven to be effective in the treatment of melioidosis may therefore be effective for treating glanders.     

Burkholderia pseudomallei animal and human isolates from Malaysia exhibit different phenotypic characteristics

Burkholderia pseudomallei is a Gram-negative saprophytic soil bacterium, which is the etiologic agent of melioidosis, a severe and fatal infectious disease occurring in human and animals. Distinct clinical and animal isolates have been shown to exhibit differences in phenotypic trait such as growth rate, colony morphology, antimicrobial resistance, and virulence. This study was carried out to gain insight into the intrinsic differences between 4 clinical and 6 animal B. pseudomallei isolates from Malaysia. The 16S rRNA-encoding genes from these 10 isolates of B. pseudomallei were sequenced to confirm the identity of these isolates along with the avirulent Burkholderia thailandensis. The nucleotide sequences indicated that the 16S rRNA-encoding genes among the 10 B. pseudomallei isolates were identical to each other. However, the nucleotide sequence differences in the 16S rRNA-encoding genes appeared to be B. pseudomallei and B. thailandensis specific. The growth rate of all B. pseudomallei isolates was determined by generating growth curves at 37 degrees C for 72 h. The isolates were found to differ in growth rates with doubling time varying from 1.5 to 2.3 h. In addition, the B. pseudomallei isolates exhibited considerable variation in colony morphology when grown on Ashdown media, brain-heart infusion agar, and Luria-Bertani agar over 9 days of observation. Antimicrobial susceptibility tests indicated that 80% of the isolates examined were Amp(R) Cb(R) Kn(R) Gm(R) Chl(S) Te(S). Virulence of the B. pseudomallei clinical and animal isolates was evaluated in B. pseudomallei-susceptible BALB/c mice. Most of the clinical isolates were highly virulent. However, virulence did not correlate with isolate origin since 2 of the animal isolates were also highly virulent.     

Burkholderia pseudomallei class a beta-lactamase mutations that confer selective resistance against ceftazidime or clavulanic acid inhibition

Burkholderia pseudomallei, the causative agent of melioidosis, is inherently resistant to a variety of antibiotics including aminoglycosides, macrolides, polymyxins, and beta-lactam antibiotics. Despite resistance to many beta-lactams, ceftazidime and beta-lactamase inhibitor-beta-lactam combinations are commonly used for treatment of melioidosis. Here, we examine the enzyme kinetics of beta-lactamase isolated from mutants resistant to ceftazidime and clavulanic acid inhibition and describe specific mutations within conserved motifs of the beta-lactamase enzyme which account for these resistance patterns. Sequence analysis of regions flanking the B. pseudomallei penA gene revealed a putative regulator gene located downstream of penA. We have cloned and sequenced the penA gene from B. mallei and found it to be identical to penA from B. pseudomallei.     

Fulminant septic melioidosis after a vacation in Thailand

Severe infection with Burkholderia pseudomallei (formerly Pseudomonas pseudomallei), the bacterium causing melioidosis, is a common cause of acquired septicaemia in south-east Asia and northern Australia. A few cases of infected travellers returning to European countries have been reported. Melioidosis is a tropical disease, the clinical presentation ranging from asymptomatic infection to fulminant sepsis. Predisposing conditions such as impaired cellular immunity, preexisting renal failure or diabetes mellitus seem to enhance the severity of the disease. For a definite diagnosis the bacterium has to be isolated. The antimicrobial treatment of choice is ceftazidime in combination with co-trimoxazole or doxycycline. Even with correct antibiotic treatment the mortality rate is high in cases of fulminant sepsis. We report a 29-year old man with Type I diabetes who acquired melioidosis during a vacation in Thailand. After returning to Austria he was admitted to the intensive care unit with multiple organ failure. Despite intensive care treatment the patient's infection proved lethal. Burkholderia pseudomallei was isolated from the blood and bronchoalveolar lavage.     

Comparative proteomic profiles and the potential markers between Burkholderia pseudomallei and Burkholderia thailandensis

Burkholderia pseudomallei is a bacterial pathogen causing the melioidosis disease, which is predominantly found in tropical areas of Southeast Asia and Northern Australia. Burkholderia thailandensis is a closely related species to B. pseudomallei but it is non-pathogenic species. In this study, we have constructed a proteome reference map of B. pseudomallei at the stationary phase of growth by using two-dimensional gel electrophoresis with a pH 4-7 immobilized pH gradient combined with matrix-assisted laser desorption ionization time of flight mass spectrometry. Approximately 550 spots could be detected by Coomassie brilliant blue G-250 staining, and 88 spots representing 77 unique proteins were identified. Eleven of the gene products were found in multiple spots indicating as isoforms. In attempt to detect distinctive expressed proteins between a virulent and a non-virulent species, the use of comparative proteomic profiles under the same condition were performed. We could identify more than 20 different spots. Twelve out of 14 spots are detected in B. pseudomallei and six proteins have been identified and indicated that they are involved in virulent characters of bacteria. Two hypothetical proteins were expressed and found only in B. pseudomallei. These proteins are potential markers to distinguish between these two species. Our study also provides a useful information of global intracellular protein expression and is a valuable starting point for analyzing a proteomic pathogenicity of the bacterial pathogen.     

Adaptation and antibiotic tolerance of anaerobic Burkholderia pseudomallei

The Gram-negative bacterium Burkholderia pseudomallei is the etiological agent of melioidosis and is remarkably resistant to most classes of antibacterials. Even after months of treatment with antibacterials that are relatively effective in vitro, there is a high rate of treatment failure, indicating that this pathogen alters its patterns of antibacterial susceptibility in response to cues encountered in the host. The pathology of melioidosis indicates that B. pseudomallei encounters host microenvironments that limit aerobic respiration, including the lack of oxygen found in abscesses and in the presence of nitric oxide produced by macrophages. We investigated whether B. pseudomallei could survive in a nonreplicating, oxygen-deprived state and determined if this physiological state was tolerant of conventional antibacterials. B. pseudomallei survived initial anaerobiosis, especially under moderately acidic conditions similar to those found in abscesses. Microarray expression profiling indicated a major shift in the physiological state of hypoxic B. pseudomallei, including induction of a variety of typical anaerobic-environment-responsive genes and genes that appear specific to anaerobic B. pseudomallei. Interestingly, anaerobic B. pseudomallei was unaffected by antibacterials typically used in therapy. However, it was exquisitely sensitive to drugs used against anaerobic pathogens. After several weeks of anaerobic culture, a significant loss of viability was observed. However, a stable subpopulation that maintained complete viability for at least 1 year was established. Thus, during the course of human infection, if a minor subpopulation of bacteria inhabited an oxygen-restricted environment, it might be indifferent to traditional therapy but susceptible to antibiotics frequently used to treat anaerobic infections.     

A review on melioidosis with special respect on molecular and immunological diagnostic techniques

Melioidosis is an 'emerging' tropical disease which causes diagnostic problems in endemic and especially in nonendemic areas e.g. Germany when imported. In the last decade, many efforts have been made to develop new molecular and immunological techniques for diagnostic use. However, an actual comprehensive review does not exist. Apart from classical microbiological procedures (microscopy, culture and biochemical identification) efforts have been made to identify Burkholderia pseudomallei using specific antibodies and PCR. The direct antigen detection can be done within a few hours leading to diagnosis days before cultural proof. ELISA and immunoblot techniques were examined for their ability to replace indirect hemagglutination and immunofluorescence test in serology. The diagnostic value of serological procedures for early detection of melioidosis is limited, however. The rapid and reliable diagnosis of melioidosis is required for an adequate onset of therapy. But no evaluated test kit based on the detection of specific antibodies, specific antigens, or on the amplification of species-specific DNA sequences is commercially available up to now. Even PCR testing--primers can easily be ordered by many gene technology companies--can not be recommended as B. pseudomallei DNA for positive controls is not available. Therefore, only microscopy and biochemical identification systems like the API 20NE can be used in the routine laboratory up to now. At this point, it has to be stressed that B. pseudomallei is a level 3 agent in many countries e.g. Germany and that only laboratories with high containment are allowed to handle it. In all cases the help of an experienced reference laboratory is adviced.     

A simple method to detect and differentiate Burkholderia pseudomallei and Burkholderia thailandensis using specific flagellin gene primers

We have previously shown that Burkholderia pseudomallei, the causative pathogen of melioidosis, may be discriminated from the closely related non-pathogenic species Burkholderia thailandensis by the presence of a 15 base pair deletion in the flagellin gene of B. thailandensis. Using specific flagellin gene primers flanking the distinctive region, PCR products of 191 and 176 bp in size were detected for B. pseudomallei and B. thailandensis, respectively. The sensitivity of detection is 20-80 colony forming units/reaction of B. pseudomallei and B. thailandensis cell suspension. To mimic the expected environmental situation, mixed populations of the two species were analyzed. The results showed that the PCR-based method could be use to distinguish the two species in a duplex reaction. In addition, we have developed a simplified method for direct PCR-based detection from soil samples. The result indicated that about 200 colonies of bacteria per reaction could be detected. This method can be applied to epidemiological studies, especially for investigating the ecological relationship between these two species in the environment.     

Rapid presumptive identification of Burkholderia pseudomallei with real-time PCR assays using fluorescent hybridization probes

Burkholderia pseudomallei (the etiologic agent of melioidosis) can cause pyogenic or granulomatous lesions in almost any organ. Septicemia has a case fatality rate of >40%. Early diagnosis and appropriate antibiotic therapy are crucial for survival, but cultivation, biochemical identification, and conventional PCR of B. pseudomallei are time consuming. We established real-time PCR assays using fluorescent hybridization probes targeting the 16S rDNA, the flagellin C (fliC) and the ribosomal protein subunit S21 (rpsU) genes. The test sensitivity and specificity were assessed with a representative panel of 39 B. pseudomallei, 9 B. mallei, 126 other Burkholderia strains of 29 species, and 45 clinically relevant non-Burkholderia organisms. The detection limit for the 16S rDNA, fliC, and rpsU assay was 40, 40, and 400 genome equivalents per reaction, however, in spiked blood samples it was 300, 300, and 3000, respectively. Specificity, positive and negative predictive value of the assays was 100%. In conclusion, we recommend the use of the 16S rDNA and/or fliC real-time PCR assays for the rapid identification of B. mallei and B. pseudomallei in positive blood cultures or from suspicious bacterial colonies.     

Modified virulence of antibiotic-induced Burkholderia pseudomallei filaments

Melioidosis is a life-threatening bacterial infection caused by Burkholderia pseudomallei. Some antibiotics used to treat melioidosis can induce filamentation in B. pseudomallei. Despite studies on the mechanism of virulence of the bacteria, the properties of B. pseudomallei filaments and their impact on virulence have not been investigated before. To understand the characteristics of antibiotic-induced filaments, we performed in vitro assays to compare several aspects of virulence between normal, nonfilamentous and filamentous B. pseudomallei. Normal, nonfilamentous B. pseudomallei could cause the lysis of monocytic cells, while filaments induced by sublethal concentrations of ceftazidime, ofloxacin, or trimethoprim show decreased lysis of monocytic cells, especially after prolonged antibiotic exposure. The motility of the filamentous bacteria was reduced compared to that of nonfilamentous bacteria. However, the filamentation was reversible when the antibiotics were removed, and the revertant bacteria recovered their motility and ability to lyse monocytic cells. Meanwhile, antibiotic resistance developed in revertant bacteria exposed to ceftazidime at the MIC. Our study highlights the danger of letting antibiotic concentration drop to the MIC or sub-MICs during antibiotic treatment of melioidosis. This could potentially give rise to a temporary reduction of bacterial virulence, only to result in bacteria that are equally virulent but more resistant to antibiotics, should the antibiotics be reduced or removed.     

Pacemaker infection secondary to burkholderia pseudomallei

Infection is a relatively rare but devastating complication of intracardiac device implantation. Burkholderia pseudomallei is the organism which causes melioidosis, an endemic and lethal infection in the tropics. We describe a case of pacemaker infection secondary to Burkholderia pseudomallei, which was treated by explantation of the device and appropriate antimicrobial therapy.     

PCR-RFLP based differentiation of Burkholderia mallei and Burkholderia pseudomallei

Burkholderia mallei and Burkholderia pseudomallei manifest a high similarity with regard to clinical syndromes, glanders and melioidosis. Phenotypic and genotypic characters are also highly similar. In an attempt to differentiate the two organisms, the molecular method was applied. This study aimed to identify the different DNA fragment in B. mallei, as compared with B. pseudomallei. The Sau3AI-digested genomic DNA patterns of B. mallei and B. pseudomallei are distinctive, especially the DNA fragments between 0.9-1.5 Kb in size. A 900-bp specific DNA fragment of B. mallei was cloned and sequenced. Using the specific DNA fragment as a probe, Southern blot hybridization was performed to differentiate the two species. The results of hybridization patterns are effective in to elucidating the genetic dissimilarities among these two Burkholderia species. Furthermore, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) digested with Sau3AI was developed to allow a more reliable and rapid identification of the two species. A 650-bp PCR-RFLP product of B. mallei was detected, while two fragments of 250 and 400-bp PCR-RFLP products of B. pseudomallei were visualized. The results suggest that the specific DNA fragment in our study should be of considerable use as a genetic marker for ensuring identification of the two species.     

Development of a polymerase chain reaction assay for the specific identification of Burkholderia mallei and differentiation from Burkholderia pseudomallei and other closely related Burkholderiaceae

Burkholderia mallei and Burkholderia pseudomallei, the etiologic agents responsible for glanders and melioidosis, respectively, are genetically and phenotypically similar and are category B biothreat agents. We used an in silico approach to compare the B. mallei ATCC 23344 and B. pseudomallei K96243 genomes to identify nucleotide sequences unique to B. mallei. Five distinct B. mallei DNA sequences and/or genes were identified and evaluated for polymerase chain reaction (PCR) assay development. Genomic DNAs from a collection of 31 B. mallei and 34 B. pseudomallei isolates, obtained from various geographic, clinical, and environmental sources over a 70-year period, were tested with PCR primers targeted for each of the B. mallei ATCC 23344-specific nucleotide sequences. Of the 5 chromosomal targets analyzed, only PCR primers designed to bimA(Bm) were specific for B. mallei. These primers were used to develop a rapid PCR assay for the definitive identification of B. mallei and differentiation from all other bacteria.     

Isolation of polymyxin B-susceptible mutants of Burkholderia pseudomallei and molecular characterization of genetic loci involved in polymyxin B resistance.

Burkholderia pseudomallei is a gram-negative bacterium that causes the disease known as melioidosis. This pathogen is endemic to Southeast Asia and northern Australia and is particularly problematic in northeastern Thailand. It has been previously reported that B. pseudomallei is resistant to the killing action of cationic antimicrobial peptides, including human neutrophil peptide, protamine sulfate, poly-L-lysine, magainins, and polymyxins. Recently, we have also found that the virulent clinical isolate B. pseudomallei 1026b is capable of replicating in media containing polymyxin B at concentrations of >100 mg/ml. In order to identify genetic loci that are associated with this particular resistance phenotype, we employed a Tn5-OT182 mutagenesis system in coordination with a replica plating screen to isolate polymyxin B-susceptible mutants. Of the 17,000 Tn5-OT182 mutants screened via this approach, five polymyxin B-susceptible mutants were obtained. Three of these mutants harbored Tn5-OT182 insertions within a genetic locus demonstrating strong homology to the lytB gene present in other gram-negative bacteria. Of the remaining two mutants, one contained a transposon insertion in a locus involved in lipopolysaccharide core biosynthesis (waaF), while the other contained an insertion in an open reading frame homologous to UDP-glucose dehydrogenase genes. Isogenic mutants were also constructed via allelic exchange and used in complementation analysis studies to further characterize the relative importance of each of the various genetic loci with respect to the polymyxin B resistance phenotype exhibited by B. pseudomallei 1026b.     

Efflux-mediated aminoglycoside and macrolide resistance in Burkholderia pseudomallei

Burkholderia pseudomallei, the causative agent of melioidosis, is intrinsically resistant to a wide range of antimicrobial agents including beta-lactams, aminoglycosides, macrolides, and polymyxins. We used Tn5-OT182 to mutagenize B. pseudomallei to identify the genes involved in aminoglycoside resistance. We report here on the identification of AmrAB-OprA, a multidrug efflux system in B. pseudomallei which is specific for both aminoglycoside and macrolide antibiotics. We isolated two transposon mutants, RM101 and RM102, which had 8- to 128-fold increases in their susceptibilities to the aminoglycosides streptomycin, gentamicin, neomycin, tobramycin, kanamycin, and spectinomycin. In addition, both mutants, in contrast to the parent, were susceptible to the macrolides erythromycin and clarithromycin but not to the lincosamide clindamycin. Sequencing of the DNA flanking the transposon insertions revealed a putative operon consisting of a resistance, nodulation, division-type transporter, a membrane fusion protein, an outer membrane protein, and a divergently transcribed regulatorprotein. Consistent with the presence of an efflux system, both mutants accumulated [3H] dihydro streptomycin, whereas the parent strain did not. We constructed an amr deletion strain, B. pseudomallei DD503, which was hypersusceptible to aminoglycosides and macrolides and which was used successfully in allelic exchange experiments. These results suggest that an efflux system is a major contributor to the inherent high-level aminoglycoside and macrolide resistance found in B. pseudomallei.     

强台风后类鼻疽发病情况及临床菌株分子特征的初步分析

目的 了解强台风袭击海南省后灾区类鼻疽病的发病情况及其临床菌株的分子特征。方法 2014年7月威马逊台风袭击海南省后,于3所大型医疗机构开展类鼻疽新发病例主动监测工作。对收集的病例绘制分布地图,并对发病情况及临床表现等进行描述性流行病学分析,采用多位点序列分型（MLST）、脉冲场凝胶电泳（PFGE）、多位点可变数目串联重复序列多态性分析（MLVA）等方法对病例临床分离株进行分子溯源及同源性比较。结果 调查期间共发现类鼻疽确诊病例16例,高于2013年同期病例数（7人）,其中12例（75.0%）于灾后4周内发病,其余4例于5～8周出现。病例均来自受灾严重的海南省北部地区,其中9例集中分布于台风中心途经的文昌-海口带状区域。临床表现以类鼻疽肺炎（75.0%）、类鼻疽败血症（68.8%）为主;病死率达50.0%。分子分型显示16株类鼻疽伯克霍尔德菌区分为12个序列型（ST）、13种PFGE带型及15个MLVA型别,除两株ST1325型菌株外来源均不相同。结论 威马逊台风过后2个月内海南省灾区出现聚集性类鼻疽病例;相关类鼻疽伯克霍尔德菌对应多个ST型,呈高度多态性,能够排除生物恐怖袭击的可能性。出现聚集性类鼻疽病例的原因及其与台风的关系尚需进一步调查。     

Burkholderia pseudomallei Isolates from Sarawak,Malaysian Borneo,Are Predominantly Susceptible to Aminoglycosides and Macrolides

Melioidosis is a potentially fatal disease caused by the saprophytic bacterium Burkholderia pseudomallei. Resistance to gentamicin is generally a hallmark of B. pseudomallei, and gentamicin is a selective agent in media used for diagnosis of melioidosis. In this study, we determined the prevalence and mechanism of gentamicin susceptibility found in B. pseudomallei isolates from Sarawak, Malaysian Borneo. We performed multilocus sequence typing and antibiotic susceptibility testing on 44 B. pseudomallei clinical isolates from melioidosis patients in Sarawak district hospitals. Whole-genome sequencing was used to identify the mechanism of gentamicin susceptibility. A novel allelic-specific PCR was designed to differentiate gentamicin-sensitive isolates from wild-type B. pseudomallei. A reversion assay was performed to confirm the involvement of this mechanism in gentamicin susceptibility. A substantial proportion (86%) of B. pseudomallei clinical isolates in Sarawak, Malaysian Borneo, were found to be susceptible to the aminoglycoside gentamicin, a rare occurrence in other regions where B. pseudomallei is endemic. Gentamicin sensitivity was restricted to genetically related strains belonging to sequence type 881 or its single-locus variant, sequence type 997. Whole-genome sequencing identified a novel nonsynonymous mutation within amrB, encoding an essential component of the AmrAB-OprA multidrug efflux pump. We confirmed the role of this mutation in conferring aminoglycoside and macrolide sensitivity by reversion of this mutation to the wild-type sequence. Our study demonstrates that alternative B. pseudomallei selective media without gentamicin are needed for accurate melioidosis laboratory diagnosis in Sarawak. This finding may also have implications for environmental sampling of other locations to test for B. pseudomallei endemicity.     

Paraplegia secondary to Burkholderia pseudomallei myelitis: a case report.

Bacterial infection is an uncommon cause of acute paraplegia. A 42-year-old Aboriginal man presented to a remote health clinic in northern Australia with myelitis associated with Burkholderia pseudomallei. He was treated with analgesia and intravenous flucloxacillin, ceftriaxone, and gentamicin and transferred to our hospital, where an urgent T12-L1 laminectomy and decompression was performed. Urine culture confirmed B. pseudomallei infection (melioidosis). Abdominopelvic computed tomography revealed left prostatic lobe and right periprostatic abscesses, which were managed conservatively. The patient was given intravenous ceftazidime (8g/d) for 2 months, followed by oral sulfamethoxazole (1600mg) and trimethoprim (320mg) twice daily for 8 weeks. Magnetic resonance imaging 3 weeks after his admission confirmed transverse myelitis. His rehabilitation was complicated by his difficulty in adjusting to disability, by urinary retention and fecal incontinence, by communication barriers, and his isolation from a culture familiar to him. He returned to his community after 15 weeks, free of infection, with T10-11 paraplegia and an indwelling catheter.