scholarly journals Rapid Filter-Based Detection and Culture of Burkholderia pseudomallei from Small Volumes of Urine

2017 ◽  
Vol 55 (9) ◽  
pp. 2698-2707 ◽  
Author(s):  
Pierre A. Michel ◽  
Christine Lascols ◽  
Jay E. Gee ◽  
Linda M. Weigel ◽  
David Sue
Keyword(s):  
Laboratory Testing ◽  
Burkholderia Pseudomallei ◽  
Dna Isolation ◽  
Urine Culture ◽  
Colony Growth ◽  
Detection Sensitivity ◽  
Culture Methods ◽  
Content Type ◽  
New Methods ◽  
Low Concentrations

ABSTRACTClinical outcomes of melioidosis patients improve when the infecting agent,Burkholderia pseudomallei, is rapidly detected and identified by laboratory testing. Detection ofB. pseudomalleiDNA or recovery of the pathogen by culture from urine can support a diagnosis of melioidosis and guide patient care. Two new methods, designated filter-capture DNA isolation (FCDI) and filter cellular recovery (FCR), were developed to increase the sensitivity of detection and recovery of viableB. pseudomalleicells from small volumes (0.45 ml) of urine. DNA from eight strains ofB. pseudomalleithat were spiked into synthetic urine at low concentrations (1 × 102CFU/ml) was detected in FCDI cell lysates using real-time PCR with greater consistency than with preparations from a QIAamp DNA Blood minikit. The FCR method showed greaterB. pseudomalleidetection sensitivity than conventional urine culture methods and resulted in typical colony growth at 24 h from as few as 1 × 102CFU/ml. In addition, the FCR method does not rely on precipitation of a urine pellet by centrifugation and requires a smaller volume of urine. The FCDI and FCR methods described here could improve time-to-results and decrease the number of negativeB. pseudomalleireports that are currently observed from urine culture as a consequence of samples containing low or variable bacterial cell concentrations.

scholarly journals Multilocus Sequence Typing and FlaA Sequencing Reveal the Genetic Stability of Campylobacter jejuni Enrichment during Coculture with Acanthamoeba polyphaga

2013 ◽  
Vol 79 (7) ◽  
pp. 2477-2479 ◽  
Author(s):  
Petra Griekspoor ◽  
Jenny Olofsson ◽  
Diana Axelsson-Olsson ◽  
Jonas Waldenström ◽  
Björn Olsen
Keyword(s):  
Campylobacter Jejuni ◽  
Genetic Stability ◽  
Environmental Samples ◽  
Molecular Techniques ◽  
Acanthamoeba Polyphaga ◽  
Culture Methods ◽  
Content Type ◽  
Conventional Culture ◽  
Low Concentrations ◽  
Genetic Specificity

ABSTRACTLow concentrations ofCampylobacter jejunicells in environmental samples make them difficult to study with conventional culture methods. Here, we show that enrichment by amoeba cocultures works well with low-concentration samples and that this method can be combined with molecular techniques without loss of genetic specificity.

scholarly journals Evaluation of Molecular Methods To Improve the Detection of Burkholderia pseudomallei in Soil and Water Samples from Laos

2015 ◽  
Vol 81 (11) ◽  
pp. 3722-3727 ◽  
Author(s):  
Michael Knappik ◽  
David A. B. Dance ◽  
Sayaphet Rattanavong ◽  
Alain Pierret ◽  
Olivier Ribolzi ◽  
...  
Keyword(s):  
Surface Water ◽  
Water Samples ◽  
Environmental Samples ◽  
Burkholderia Pseudomallei ◽  
Molecular Detection ◽  
Culture Methods ◽  
Environmental Distribution ◽  
Content Type ◽  
Culture Techniques ◽  
Enrichment Step

ABSTRACTBurkholderia pseudomalleiis the cause of melioidosis, a severe and potentially fatal disease of humans and animals. It is endemic in northern Australia and Southeast Asia and is found in soil and surface water. The environmental distribution ofB. pseudomalleiworldwide and within countries where it is endemic, such as the Lao People's Democratic Republic (Laos), remains unclear. However, this knowledge is important to our understanding of the ecology and epidemiology ofB. pseudomalleiand to facilitate public health interventions. Sensitive and specific methods to detectB. pseudomalleiin environmental samples are therefore needed. The aim of this study was to compare molecular and culture-based methods for the detection ofB. pseudomalleiin soil and surface water in order to identify the optimal approach for future environmental studies in Laos. Molecular detection by quantitative real-time PCR (qPCR) was attempted after DNA extraction directly from soil or water samples or after an overnight enrichment step. The positivity rates obtained by qPCR were compared to those obtained by different culture techniques. The rate of detection from soil samples by qPCR following culture enrichment was significantly higher (84/100) than that by individual culture methods and all culture methods combined (44/100;P< 0.001). Similarly, qPCR following enrichment was the most sensitive method for filtered river water compared with the sensitivity of the individual methods and all individual methods combined. In conclusion, molecular detection following an enrichment step has proven to be a sensitive and reliable approach forB. pseudomalleidetection in Lao environmental samples and is recommended as the preferred method for future surveys.

scholarly journals Urinary Microbiome Characteristics in Female Patients with Acute Uncomplicated Cystitis and Recurrent Cystitis

2021 ◽  
Vol 10 (5) ◽  
pp. 1097
Author(s):  
Jeong-Ju Yoo ◽  
Hee Shin ◽  
Ju Song ◽  
Minjung Kim ◽  
Jina Yun ◽  
...  
Keyword(s):  
Urine Culture ◽  
Culture Methods ◽  
Microbiome Composition ◽  
Additional Information ◽  
Transurethral Catheter ◽  
Urinary Microbiome ◽  
Acute Uncomplicated Cystitis ◽  
The Difference ◽  
Uncomplicated Cystitis ◽  
Recurrent Cystitis

Traditionally, the diagnostic mainstay of recurrent urinary tract infection has been urinary culture. However, the causative uropathogen of recurrent cystitis has not been well established. Urine DNA next-generation sequencing (NGS) can provide additional information on these infections. Herein, we compared urine NGS results and urine cultures in patients with acute uncomplicated cystitis (AUC) and recurrent cystitis (RC), and evaluated the difference in microbiome patterns in the NGS results. Patients who underwent urine culture and NGS due to AUC or RC were retrospectively reviewed. All urine samples were collected via a transurethral catheter and studied utilizing a type of NGS called 16S ribosomal RNA gene amplification and sequencing. The sensitivity of urine NGS was significantly higher than that of conventional urine culture (69.0% vs. 16.7%, p < 0.05). The detection rate of urine NGS was slightly lower in the RC group than in the AUC group (67.7% vs. 72.7%). Microbiome diversity was significantly higher in the RC group compared to the AUC group (p = 0.007), and the microbiome composition was significantly different between the AUC and RC groups. In the urine NGS results, Pseudomonas, Acinetobacter, and Enterobacteriaceae were found in the AUC group, and Sphingomonas, Staphylococcus, Streptococcus, and Rothia spp. were detected in the RC group. Urine NGS can significantly increase the diagnostic sensitivity compared to traditional urine culture methods, especially in RC patients. AUC and RC patients had significant differences in bacterial diversity and patterns. Therefore, recurrent cystitis might be approached from a different perspective.

scholarly journals Neutrophil Extracellular Traps Exhibit Antibacterial Activity against Burkholderia pseudomallei and Are Influenced by Bacterial and Host Factors

2012 ◽  
Vol 80 (11) ◽  
pp. 3921-3929 ◽  
Author(s):  
Donporn Riyapa ◽  
Surachat Buddhisa ◽  
Sunee Korbsrisate ◽  
Jon Cuccui ◽  
Brendan W. Wren ◽  
...  
Keyword(s):  
Burkholderia Pseudomallei ◽  
Capsular Polysaccharide ◽  
Neutrophil Extracellular Traps ◽  
Polymorphonuclear Neutrophils ◽  
Dependent Manner ◽  
Predisposing Factor ◽  
Bacterial Killing ◽  
Content Type ◽  
Extracellular Traps ◽  
Type Iii Protein Secretion

ABSTRACTBurkholderia pseudomalleiis the causative pathogen of melioidosis, of which a major predisposing factor is diabetes mellitus. Polymorphonuclear neutrophils (PMNs) kill microbes extracellularly by the release of neutrophil extracellular traps (NETs). PMNs play a key role in the control of melioidosis, but the involvement of NETs in killing ofB. pseudomalleiremains obscure. Here, we showed that bactericidal NETs were released from human PMNs in response toB. pseudomalleiin a dose- and time-dependent manner.B. pseudomallei-induced NET formation required NADPH oxidase activation but not phosphatidylinositol-3 kinase, mitogen-activated protein kinases, or Src family kinase signaling pathways.B. pseudomalleimutants defective in the virulence-associated Bsa type III protein secretion system (T3SS) or capsular polysaccharide I (CPS-I) induced elevated levels of NETs. NET induction by such mutants was associated with increased bacterial killing, phagocytosis, and oxidative burst by PMNs. Taken together the data imply that T3SS and the capsule may play a role in evading the induction of NETs. Importantly, PMNs from diabetic subjects released NETs at a lower level than PMNs from healthy subjects. Modulation of NET formation may therefore be associated with the pathogenesis and control of melioidosis.

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

2017 ◽  
Vol 61 (6) ◽  
Author(s):  
Julia V. Bugrysheva ◽  
David Sue ◽  
Jay E. Gee ◽  
Mindy G. Elrod ◽  
Alex R. Hoffmaster ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Clavulanic Acid ◽  
Burkholderia Pseudomallei ◽  
Efflux Pump ◽  
Point Mutations ◽  
Comparative Genomic ◽  
Content Type ◽  
Reductase Gene ◽  
Amoxicillin Clavulanic Acid ◽  
Imipenem Resistance

ABSTRACT Burkholderia pseudomallei Bp1651 is resistant to several classes of antibiotics that are usually effective for treatment of melioidosis, including tetracyclines, sulfonamides, and β-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 β-lactamase and was previously implicated in resistance to β-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.

scholarly journals Hormetic Concentrations of Hydrogen Peroxide but Not Ethanol Induce Cross-Adaptation to Different Stresses in Budding Yeast

2014 ◽  
Vol 2014 ◽  
pp. 1-5 ◽  
Author(s):  
Halyna M. Semchyshyn
Keyword(s):  
Hydrogen Peroxide ◽  
Dose Response ◽  
Budding Yeast ◽  
Regulatory Protein ◽  
Colony Growth ◽  
Inhibitory Effect ◽  
Cross Resistance ◽  
Mild Stress ◽  
Low Concentrations ◽  
Cross Adaptation

The biphasic-dose response of microorganisms to hydrogen peroxide is a phenomenon of particular interest in hormesis research. In different animal models, the dose-response curve for ethanol is also nonlinear showing an inhibitory effect at high doses but a stimulatory effect at low doses. In this study, we observed the hormetic-dose response to ethanol in budding yeastS. cerevisiae. Cross-protection is a phenomenon in which exposure to mild stress results in the acquisition of cellular resistance to lethal stress induced by different factors. Since both hydrogen peroxide and ethanol at low concentrations were found to stimulate yeast colony growth, we evaluated the role of one substance in cell cross-adaptation to the other substance as well as some weak organic acid preservatives. This study demonstrates that, unlike ethanol, hydrogen peroxide at hormetic concentrations causes cross-resistance ofS. cerevisiaeto different stresses. The regulatory protein Yap1 plays an important role in the hormetic effects by low concentrations of either hydrogen peroxide or ethanol, and it is involved in the yeast cross-adaptation by low sublethal doses of hydrogen peroxide.

scholarly journals Correlates of Immune Protection following Cutaneous Immunization with an Attenuated Burkholderia pseudomallei Vaccine

2013 ◽  
Vol 81 (12) ◽  
pp. 4626-4634 ◽  
Author(s):  
Ediane B. Silva ◽  
Andrew Goodyear ◽  
Marjorie D. Sutherland ◽  
Nicole L. Podnecky ◽  
Mercedes Gonzalez-Juarrero ◽  
...  
Keyword(s):  
Immune Responses ◽  
Vaccine Strain ◽  
Protective Immunity ◽  
Burkholderia Pseudomallei ◽  
Partial Protection ◽  
Content Type ◽  
Humoral Immune ◽  
Humoral Immune Responses ◽  
Immune Correlates ◽  
Draining Lymph Nodes

ABSTRACTInfections with the Gram-negative bacteriumBurkholderia pseudomallei(melioidosis) are associated with high mortality, and there is currently no approved vaccine to prevent the development of melioidosis in humans. Infected patients also do not develop protective immunity to reinfection, and some individuals will develop chronic, subclinical infections withB. pseudomallei. At present, our understanding of what constitutes effective protective immunity againstB. pseudomalleiinfection remains incomplete. Therefore, we conducted a study to elucidate immune correlates of vaccine-induced protective immunity against acuteB. pseudomalleiinfection. BALB/c and C57BL/6 mice were immunized subcutaneously with a highly attenuated, Select Agent-excludedpurMdeletion mutant ofB. pseudomallei(strain Bp82) and then subjected to intranasal challenge with virulentB. pseudomalleistrain 1026b. Immunization with Bp82 generated significant protection from challenge withB. pseudomallei, and protection was associated with a significant reduction in bacterial burden in lungs, liver, and spleen of immunized mice. Humoral immunity was critically important for vaccine-induced protection, as mice lacking B cells were not protected by immunization and serum from Bp82-vaccinated mice could transfer partial protection to nonvaccinated animals. In contrast, vaccine-induced protective immunity was found to be independent of both CD4 and CD8 T cells. Tracking studies demonstrated uptake of the Bp82 vaccine strain predominately by neutrophils in vaccine-draining lymph nodes and by smaller numbers of dendritic cells (DC) and monocytes. We concluded that protection following cutaneous immunization with a live attenuatedBurkholderiavaccine strain was dependent primarily on generation of effective humoral immune responses.

Improved diagnosis of gastrointestinal infections using a semi-automated multiplex real-time PCR for detection of enteropathogens

2021 ◽  
Vol 70 (9) ◽  
Author(s):  
Berta Fidalgo ◽  
Elisa Rubio ◽  
Victor Pastor ◽  
Marta Parera ◽  
Clara Ballesté-Delpierre ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Enteric Pathogens ◽  
Pcr Analysis ◽  
Culture Methods ◽  
Gastrointestinal Infections ◽  
Content Type ◽  
Link Type ◽  
Microbiological Culture ◽  
Micro Organisms

Introduction. The identification of enteropathogens is critical for the clinical management of patients with suspected gastrointestinal infection. The FLOW multiplex PCR system (FMPS) is a semi-automated platform (FLOW System, Roche) for multiplex real-time PCR analysis. Hypothesis/Gap Statement. FMPS has greater sensitivity for the detection of enteric pathogens than standard methods such as culture, biochemical identification, immunochromatography or microscopic examination. Aim.The diagnostic performance of the FMPS was evaluated and compared to that of traditional microbiological procedures. Methodology. A total of 10 659 samples were collected and analysed over a period of 7 years. From 2013 to 2018 (every July to September), samples were processed using standard microbiological culture methods. In 2019, the FMPS was implemented using real-time PCR to detect the following enteropathogens: Shigella spp., Salmonella spp., Campylobacter spp., Giardia intestinalis, Entamoeba histolytica, Blastocystis hominis, Cryptosporidum spp., Dientamoeba fragilis, adenovirus, norovirus and rotavirus. Standard microbiological culture methods (2013–2018) included stool culture, microscopy and immunochromatography. Results. A total of 1078 stool samples were analysed prospectively using the FMPS from July to September (2019): bacterial, parasitic and viral pathogens were identified in 15.3, 9.71 and 5.29 % of cases, respectively. During the same period of 6 years (2013–2018), the proportion of positive identifications using standard microbiological methods from 2013 to 2018 was significantly lower. A major significant recovery improvement was observed for all bacteria species tested: Shigella spp./enteroinvasive Escherichia coli (EIEC) (P <0.05), Salmonella spp. (P <0.05) and Campylobacter spp. (P <0.05). Marked differences were also observed for the parasites G. intestinalis, Cryptosporidium spp. and D. fragilis. Conclusion. These results support the value of multiplex real-time PCR analysis for the detection of enteric pathogens in laboratory diagnosis with outstanding performance in identifying labile micro-organisms. The identification of unsuspected micro-organisms for less specific clinical presentations may also impact on clinical practice and help optimize patient management.

scholarly journals Regulatory Effect of SlyA onrcsBExpression inSalmonella entericaSerovar Typhimurium

2018 ◽  
Vol 201 (4) ◽  
Author(s):  
María F. Ballesteros ◽  
Mónica F. Torrez Lamberti ◽  
Juan V. Farizano ◽  
María M. Pescaretti ◽  
Mónica A. Delgado
Keyword(s):  
Virulence Gene ◽  
Content Type ◽  
Virulence Gene Expression ◽  
Low Concentrations ◽  
Antagonistic Behavior ◽  
Serovar Typhimurium ◽  
Direct Binding ◽  
High Concentrations ◽  
Encoding Genes ◽  
Different Levels

ABSTRACTTheSalmonella entericaserovar Typhimurium RcsCDB system regulates the synthesis of colanic acid and the flagellum as well as the expression of virulence genes. We previously demonstrated that thercsC11mutant, which constitutively activates the RcsB regulator, attenuatesSalmonellavirulence in an animal model. This attenuated phenotype was also produced by deletion of theslyAgene. In this work, we investigated if this antagonistic behavior is produced by modulating the expression of both regulator-encoding genes. We demonstrated that SlyA overproduction negatively regulatesrcsBtranscription. A bioinformatics analysis enabled us to identify putative SlyA binding sites on both promoters, PrcsDBand PrcsB, which controlrcsBtranscriptional levels. We also determined that SlyA is able to recognize and bind to these predicted sites to modulate the activity of bothrcsBpromoters. According to these results, SlyA repressesrcsBtranscription by direct binding to specific sites located on thercsBpromoters, thus accounting for the attenuated/virulence antagonistic behaviors. Moreover, we showed that the opposite effect between both regulators also physiologically affects theSalmonellamotility phenotype. In this sense, we observed that under SlyA overproduction, PrcsBis repressed, and consequently, bacterial motility is increased. On the basis of these results, we suggest that during infection, the different RcsB levels produced act as a switch between the virulent and attenuated forms ofSalmonella. Thereby, we propose that higher concentrations of RcsB tilt the balance toward the attenuated form, while absence or low concentrations resulting from SlyA overproduction tilt the balance toward the virulent form.IMPORTANCEThe antagonistic behavior of RcsB and SlyA on virulence gene expression led us to hypothesize that there is interplay between both regulators in a regulatory network and these could be considered coordinators of this process. Here, we report that the SlyA virulence factor influences motility behavior by controllingrcsBtranscription from the PrcsBpromoter. We also demonstrate that SlyA negatively affects the expression of thercsBgene by direct binding to PrcsDBand PrcsBpromoters. We suggest that different levels of RcsB act as a switch between the virulent and attenuated forms ofSalmonella, where high concentrations of the regulator tend to tilt the balance toward the attenuated form and low concentrations or its absence tilt it toward the virulent form.

scholarly journals Whole-genome comparative analysis of Malaysian Burkholderia pseudomallei clinical isolates

2021 ◽  
Vol 7 (2) ◽  
Author(s):  
Ahmad-Kamal Ghazali ◽  
Su-Anne Eng ◽  
Jia-Shiun Khoo ◽  
Seddon Teoh ◽  
Chee-Choong Hoh ◽  
...  
Keyword(s):  
Comparative Analysis ◽  
Virulence Factors ◽  
Type Species ◽  
Burkholderia Pseudomallei ◽  
Clinical Manifestations ◽  
Phylogenomic Analysis ◽  
Content Type ◽  
Link Type ◽  
Multiple Organs ◽  
The Difference

Burkholderia pseudomallei , a soil-dwelling Gram-negative bacterium, is the causative agent of the endemic tropical disease melioidosis. Clinical manifestations of B. pseudomallei infection range from acute or chronic localized infection in a single organ to fulminant septicaemia in multiple organs. The diverse clinical manifestations are attributed to various factors, including the genome plasticity across B. pseudomallei strains. We previously characterized B. pseudomallei strains isolated in Malaysia and noted different levels of virulence in model hosts. We hypothesized that the difference in virulence might be a result of variance at the genome level. In this study, we sequenced and assembled four Malaysian clinical B. pseudomallei isolates, UKMR15, UKMPMC2000, UKMD286 and UKMH10. Phylogenomic analysis showed that Malaysian subclades emerged from the Asian subclade, suggesting that the Malaysian strains originated from the Asian region. Interestingly, the low-virulence strain, UKMH10, was the most distantly related compared to the other Malaysian isolates. Genomic island (GI) prediction analysis identified a new island of 23 kb, GI9c, which is present in B. pseudomallei and Burkholderia mallei , but not Burkholderia thailandensis . Genes encoding known B. pseudomallei virulence factors were present across all four genomes, but comparative analysis of the total gene content across the Malaysian strains identified 104 genes that are absent in UKMH10. We propose that these genes may encode novel virulence factors, which may explain the reduced virulence of this strain. Further investigation on the identity and role of these 104 proteins may aid in understanding B. pseudomallei pathogenicity to guide the design of new therapeutics for treating melioidosis.

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