Infection by Ralstonia species in cystic fibrosis patients: identification of R. pickettii and R. mannitolilytica by polymerase chain reaction. (Research).The frequency of respiratory tract infections caused by Ralstonia species in persons with cystic fibrosis cystic fibrosis (sĭs`tĭk fībrō`sĭs), inherited disorder of the exocrine glands (see gland), affecting children and young people; median survival is 25 years in females and 30 years in males. (CF) and the role of these species in CF pulmonary disease are not well documented. In part, this lack of documentation may be attributed to the difficulty in accurately identifying Ralstonia species; R. mannitolilytica and R. pickettii in particular may be misidentified as other closely related species, particularly those of the Burkholdeda cepacia complex. We used polyphasic analyses to identify 42 Ralstonia isolates from sputum cultures from 38 CF patients. Several isolates that could not be identified to the species level may belong to novel Ralstonia species. We demonstrated chronic colonization by using genotyping of serial isolates recovered from the same patient. To facilitate identification of R. mannitolilytica and R. pickettii, we developed 16S ribosomal DNA-based polymerase chain reaction polymerase chain reaction (pŏl`ĭmərās') (PCR), laboratory process in which a particular DNA segment from a mixture of DNA chains is rapidly replicated, producing a large, readily analyzed sample of a piece of DNA; the process is assays that allow sensitive and specific identification of these species.
Cystic fibrosis (CF) is the most frequent hereditary disease in Caucasian populations (1); chronic microbial microbial
pertaining to or emanating from a microbe.
the breakdown of organic material, especially feedstuffs, by microbial organisms. colonization of the large airways, leading to exacerbations of pulmonary infection, is the major cause of illness and death in CF patients. Typical CF pathogens include Staphylococcus aureus Staphylococcus au·re·us
A bacterium that causes furunculosis, pyemia, osteomyelitis, suppuration of wounds, and food poisoning.
Staphylococcus aureus Staphylococcus pyogenes , Pseudornonas aeruginosa, Haemophilus influenzae Haemophilus in·flu·en·zae
A gram-negative, rod-shaped bacterium of the genus Haemophilus, especially Haemophilus influenzae type b, that occurs in the human respiratory tract and causes acute respiratory infections, acute conjunctivitis, and , and Burkholderia cepacia complex Burkholderia cepacia complex (BCC), or simply Burkholderia cepacia is a group of catalase-producing, non-lactose-fermenting Gram-negative bacteria composed of at least nine different species, including B. cepacia, B. multivorans, B. ; other species, including Stenotrophornonas maltophilia, Alcaligenes (Achromobacter) xylosoxidans, B. gladioli glad·i·o·lus
n. pl. glad·i·o·li or glad·i·o·lus·es
1. also glad·i·o·la Botany Any of numerous plants of the genus Gladiolus, , and R. pickettii have been recovered from sputum cultures of CF patients as well (2,3). Recently, we showed that a number of unusual bacterial species (including several novel species within the [alpha]-Proteobacteria) are also occasionally isolated from CF patients (4). Infection with mucoid mucoid /mu·coid/ (mu´koid)
1. resembling mucus.
Any of various glycoproteins similar to the mucins, especially a mucoprotein.
adj. P. aeruginosa and members of the B. cepacia complex is associated with increased illness and death in CF patients (5-7), but the clinical importance of infection with these other species is less clear.
The genus Ralstonia was proposed in 1995 (8). Since its creation, the taxonomy of the genus has expanded to include 11 species, which are R. pickettii, R. solanacearum, R. eutropha, R. gilardii, R. paucula, R. basilensis, R. oxalatica, R. mannitolilytica, R. taiwanensis, R. campinensis, and R. metallidurans (8-14). Ralstonia spp. are isolated from a wide variety of ecologic niches, including plants and soils contaminated contaminated,
v 1. made radioactive by the addition of small quantities of radioactive material.
2. made contaminated by adding infective or radiographic materials.
3. an infective surface or object. with heavy metals heavy metals,
n.pl metallic compounds, such as aluminum, arsenic, cadmium, lead, mercury, and nickel. Exposure to these metals has been linked to immune, kidney, and neurotic disorders. . R. pickettii has been associated with nosocomial nosocomial /noso·co·mi·al/ (nos?o-ko´me-il) pertaining to or originating in a hospital.
1. Of or relating to a hospital.
2. outbreaks caused by contaminated solutions used for patient care and with pseudoepidemics caused by contaminated solutions in the diagnostic laboratory (15-21). Several hospital-associated outbreaks attributed to R. mannitolilytica (formerly known as R. pickettii biovar 3 or P thornasii) have been described (12,22,23). R. paucula and R. gilardii have only sporadically been isolated from human clinical samples, including cerebrospinal fluid cerebrospinal fluid (CSF)
Clear, colourless liquid that surrounds the brain and spinal cord and fills the spaces in them. It helps support the brain, acts as a lubricant, maintains pressure in the skull, and cushions shocks. , bone marrow, wounds, and the respiratory tract respiratory tract
The air passages from the nose to the pulmonary alveoli, including the pharynx, larynx, trachea, and bronchi.
Respiratory tract (9,10). A complete assessment of the frequency of human infection due to Ralstonia species is confounded by the difficulty in accurate species identification by using standard microbiologic techniques. Indeed, these species are frequently misidentified as P fluorescens or B. cepacia complex (12,24-26).
We describe the occurrence of several Ralstonia species in the respiratory secretions of CF patients. We also describe the development and evaluation of two polymerase chain reaction (PCR PCR polymerase chain reaction.
polymerase chain reaction
Polymerase chain reaction (PCR) ) assays for rapid, accurate identification of R. pickettii and R. mannitolilytica.
Materials and Methods
Bacterial Strains and Study Population Since early 1997, the Burkholderia cepacia Burkholderia cepacia Pseudomonas cepacia Bacteriology A bacterium found in the environment–eg, plants, water, soil, and in hospital environment, which may colonize the respiratory tract of Pts with cystic fibrosis; transmitted by direct physical Research Laboratory and Repository (University of Michigan (body, education) University of Michigan - A large cosmopolitan university in the Midwest USA. Over 50000 students are enrolled at the University of Michigan's three campuses. The students come from 50 states and over 100 foreign countries. , Ann Arbor Ann Arbor, city (1990 pop. 109,592), seat of Washtenaw co., S Mich., on the Huron River; inc. 1851. It is a research and educational center, with a large number of government and industrial research and development firms, many in high-technology fields such as , MI) has received more than 4,000 bacterial isolates, collected from CF patients receiving care in 145 CF treatment centers in 130 U. S. cities. Isolates received were tentatively identified by the referring microbiology laboratory as B. cepacia complex or a related species or were not identified to the species level. From these isolates, we identified 42 Ralstonia isolates obtained from 38 patients who had received care in 19 treatment centers in 18 U. S. cities. The type and reference strains of Ralstonia, Pandoraea, Burkholderia, Alcaligenes, and Bordetella Bordetella
A genus of gram-negative bacteria which are coccobacilli and obligate aerobes, and fail to ferment carbohydrates. These bacteria are respiratory pathogens. Bordetella pertussis, B. parapertussis, and B. species have been described (9-14). These strains were obtained from the BCCM/LMG-Bacteria Collection (Laboratorium voor Microbiologie, Universiteit Gent, Belgium) or were provided by D. Henry (University of British Columbia Locations
The Vancouver campus is located at Point Grey, a twenty-minute drive from downtown Vancouver. It is near several beaches and has views of the North Shore mountains. The 7. , Vancouver, Canada). All isolates were grown aerobically on Mueller-Hinton broth (Becton, Dickinson and Company, Cockeysville, MD) supplemented with 1.8% (wt/vol) agar and incubated at 32[degrees]C.
We used a polyphasic approach to identify all isolates, including biochemical tests, 16S ribosomal (r)DNA-based PCR assays and sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel electrophoresis
Electrophoresis performed in a gel composed of agarose, polyacrylamide, or starch. (SDS-PAGE SDS-PAGE
sodium dodecyl sulfate-polyacrylamide gel electrophoresis. ) of whole-cell proteins. Biochemical tests (determination of oxidase oxidase /ox·i·dase/ (ok´si-das) any enzyme of the class of oxidoreductases in which molecular oxygen is the hydrogen acceptor.
n. , lysine decarboxylase Lysine decarboxylase is an enzyme which converts lysine to cadaverine. External links
• • , and o-nitrophenyl-[beta]-D-galactoside activity; growth on B. cepacia selective agar; and oxidation-fermentation of sucrose) were performed as described (27). SDS-PAGE of whole-cell proteins was performed as described (9,10), and isolates were identified by comparison with a database containing protein profiles of all Ralstonia species. We used 16S rDNA-based PCR assays (28) to determine whether or not isolates belonged to the genera Burkholderia or Ralstonia or to the B. cepacia complex.
Genotyping of Serial Isolate
Multiple isolates from a single patient were genotyped by randomly amplified polymorphic polymorphic - polymorphism DNA DNA: see nucleic acid.
or deoxyribonucleic acid
One of two types of nucleic acid (the other is RNA); a complex organic compound found in all living cells and many viruses. It is the chemical substance of genes. (RAPD RAPD Randomly Amplified Polymorphic DNA
RAPD relative afferent pupillary defect (ophthalmology; aka Marcus-Gunn Pupil) ) genotyping as described (29). We digitized gel images with a GelDoc2000 gel analyzer (Bio-Rad Laboratories, Hercules, CA) and stored them as tagged image files. After normalization In relational database management, a process that breaks down data into record groups for efficient processing. There are six stages. By the third stage (third normal form), data are identified only by the key field in their record. with the molecular weight marker, patterns were analyzed with Molecular Analyst Fingerprinting Plus software (Bio-Rad Laboratories). Similarities between patterns were calculated by using Pearson's product-moment correlation coefficient Noun 1. product-moment correlation coefficient - the most commonly used method of computing a correlation coefficient between variables that are linearly related
Pearson product-moment correlation coefficient . We considered isolates to belong to the same genotype if they shared 90% or more similarity.
Development of Primers for Species-Specific PCR Assays
We retrieved 16S rDNA sequences of all Ralstonia spp. and representatives of related genera from the GenBank database, using the MegAlign software package (DNASTAR Inc., Madison, WI) to align the sequences. Based on this alignment, we developed primers specific for R. pickettii and R. mannitolilytica: Rp-F1 (5'-ATGATCTAGCTTGCTAGATTGAT-3') and Rp-R1 (5'-ACTGATCGTCGCCTTGGTG-3') (forward and reverse primers for the identification of R. pickettii) and Rm-F1 (5'-GGGAAAGCTTGCTTTCCTGCC-3') and Rm-R1 (5'-TCCGGGTATTAACCAGAGCCAT-3') (forward and reverse primers for the identification of R. mannitolilytica).
Polymerase Chain Reaction
DNA was prepared as described (30). PCR assays were performed in 25-[micro]L reaction mixtures, containing 2 [micro]L DNA solution, 1U Taq polymerase Taq polymerase ("Taq Pol," or simply "Taq") is a thermostable polymerase used in polymerase chain reaction to check for the presence or absence of a gene by amplifying a DNA fragment. It replaced E.coli DNA polymerase in PCR because of the temperature conditions of PCR. (GIBCO GIBCO Grand Island Biological Company (tissue culture media enterprise) Invitrogen Corp., Gaithersburg, MD), 250 mM (each) deoxynucleotide triphosphate triphosphate /tri·phos·phate/ (tri-fos´fat) a salt containing three phosphate radicals.
A salt or ester containing three phosphate groups. (GIBCO Invitrogen Corp.), 1.5 mM Mg[Cl.sub.2], 1x PCR buffer (GIBCO Invitrogen Corp.), and 20 pmol of each oligonucleotide primer. Amplification was carried out with a PTC-100 programmable thermal cycler The Thermal cycler (also known as a thermocycler, PCR machine or DNA amplifier) is a laboratory apparatus used for PCR. The device has a thermal block with holes where tubes with the PCR reaction mixtures can be inserted. (Labtrade Inc., Miami, FL). After initial denaturation denaturation, term used to describe the loss of native, higher-order structure of protein molecules in solution. Most globular proteins exhibit complicated three-dimensional folding described as secondary, tertiary, and quarternary structures. for 2 min at 94[degrees]C, 30 amplification cycles were completed, each consisting of 1 min at 94[degrees]C, 1 min at 55[degrees]C (for identifying R. pickettii) or 57[degrees]C (for identifying R. mannitolilytica), and 1 min 30 s at 72[degrees]C. A final extension of 10 min at 72[degrees]C was applied. Negative control PCRs with all reaction mixture components except template DNA were used for every experiment.
Evaluation of the PCR Assays
For evaluating PCR assays, we tested 152 isolates, including 79 Ralstonia isolates (both clinical isolates and reference strains) and 73 isolates representing phylogenetically phy·lo·ge·net·ic
1. Of or relating to phylogeny or phylogenetics.
2. Relating to or based on evolutionary development or history: a phylogenetic classification of species. related species that may be found in sputum cultures of CF patients. Isolates tested were as follows: R. pickettii (27 isolates), R. mannitolilytica (34), R. gilardii (4), R. paucula (2), R. taiwanensis (1), R. basilensis (1), R. eutropha (1), R. oxalatica (1), R. solanacearum (1), R. campinensis (1), R. metallidurans (1), Ralstonia sp. (5), B. cepacia genomovar I (3), B. multivorans (2), B. cepacia genomovar III (7), B. stabilis (2), B. vietnamiensis (2), B. cepacia genomovar VI (5), B. ambifaria (3), B. gladioli (6), B. fungorum (1), Pandoraea apista (5), P. norimbergensis (3), P. pnomenusa (2), P. sputorum (4), P. pulmonicola (2), Alcaligenes xylosoxidans (5), P. aeruginosa (5), S. maltophilia (5), and one isolate each of A. denitrificans, A. piechaudii, A. faecalis, A. ruhlandii, Bordetella avium, B. hinzii, B. trematum, B. bronchiseptica, B. pertussis pertussis: see whooping cough. , B. parapertussis, and B. holmesii.
Isolates were tentatively identified as belonging to the genus Ralstonia if they 1) reacted with primer pair RHG-F/ RHG-R (specific for Burkholderia and Ralstonia spp.) (28), 2) showed no lysine decarboxylase and o-nitrophenyl-[beta]-D-galactoside activity, 3) produced no acid from sucrose, and 4) showed oxidase activity. Using these criteria, we identified 42 putative Ralstonia sp. isolates. These isolates were further identified to the species level by using SDS-PAGE of whole-cell proteins. Most isolates (25) were identified as R. mannitolilytica; 9 were identified as R. pickettii. Two isolates were identified as R. gilardii, and another as R. taiwanensis. Five isolates clearly belonged to the genus Ralstonia but could not be classified into one of the known species. Pending further investigations, these isolates were classified as Ralstonia sp.
Genotyping of Serial Isolates
We identified two patients (A and B) who were sputum-culture positive for R. mannitolilytica and one patient (C) who was culture positive for R. pickettii on more than one occasion. The three R. mannitolilytica isolates cultured from patient A were recovered over a period of >2 years. RAPD genotyping indicated that the first isolate clearly differed from the two isolates recovered subsequently; the latter two isolates (recovered 20 months apart) were the same genotype (Figure 1). Similarly, the two R. mannitolilytica isolates recovered from patient B (cultured 8 weeks apart) were the same genotype, as were the two R. pickettii isolates recovered from patient C (cultured 6 weeks apart) (Figure 1).
[FIGURE 1 OMITED]
Alignment of 16S rRNA gene sequences of Ralstonia sp. available in GenBank showed similarity values [greater than or equal to] 3.1% and [greater than or equal to] 98.2% within the species R. pickettii and R. mannitolilytica, respectively. Identity of sequences between these two species ranged from 89.9% to 96.8%. Several species-level sequence signatures were detected and were incorporated into the species-specific primers Rp-F1 and Rp-R1 (forward and reverse primer for R. pickettii) and Rm-F1 and Rm-R1 (forward and reverse primer for R. mannitolilytica). PCR with these primers resulted in the amplification of fragments of 210 bp and 398 bp, respectively (Figure 2). Each of the 152 strains included in this study was examined by PCR with the primer pairs described (Table).
[FIGURE 2 OMITTED]
The occurrence and clinical role of Ralstonia sp. in the respiratory secretions of persons with CF have not been systematically investigated because of the rapidly changing taxonomy of the genus Ralstonia and the absence of rapid, reliable methods for species identification. We used a polyphasic approach to identify Ralstonia sp. in sputum cultures of CF patients and developed two PCR assays for identifying R. pickettii and R. mannitolilytica.
Previous reports describing the bacterial flora The bacterial flora is the whole system of bacteria in body cavities that have contact with the outside world. Every place shows another biochemical environment:
Our data do not provide evidence for patient-to-patient spread of Ralstonia sp. because no clustering of cases occurred within centers or geographic regions (data not shown). However, we were able to document persistent colonization with Ralstonia species in three patients. Patient A's infection is particularly interesting. In this patient, an initial R. mannitolilytica strain was apparently replaced with another strain, which then persisted for >20 months. However, the bacterial and host factors involved in infection by more than one R. mannitolilytica strain or with chronic colonization remain to be defined.
Five Ralstonia isolates could not be identified to the species level. 16S rDNA PCR and SDS-PAGE of whole-cell proteins clearly indicated that these isolates belong to the genus Ralstonia, suggesting that they may represent novel Ralstonia sp. Further polyphasic taxonomic studies are needed to clarify their status. The finding of R. mannitolilytica, R. gilardii, R. taiwanensis, and possible novel Ralstonia species in respiratory secretions of CF patients suggests that these organisms may be emerging human pathogens and again highlights the fact that the bacterial biodiversity in the respiratory tract of CF patients has thus far been underestimated (4).
Of the 25 R. mannitolilytica strains identified in this study, 9 were initially identified by the referring laboratory as R. pickettii, 8 as B. cepacia complex, 6 as Burkholderia sp., 1 as B. gladioli, and 1as P fluorescens. Of the 9 R. pickettii strains identified, 3 were identified by the referring laboratory as R. pickettii, 2 as Burkholderia sp., 1 as Pseudomonas Pseudomonas
A genus of gram-negative, nonsporeforming, rod-shaped bacteria. Motile species possess polar flagella. They are strictly aerobic, but some members do respire anaerobically in the presence of nitrate. sp., 1 as B. cepacia complex, and 2 isolates as unidentified. The R. gilardii and R. taiwanensis isolates were received as B. cepacia complex and S. maltophilia, respectively. Most (81%) of these isolates were capable of growth on B. cepacia selective agar. These observations reiterate that identification of these species is not straightforward and that their misidentification as other CF pathogens, such as B. cepacia complex, is not uncommon. Such misidentification has an important impact on infection control in CF since the efficiency of these measures depends on accurate identification of the microorganisms involved. Infection-control policies, particularly those recommended to prevent interpatient spread of B. cepacia complex, have a tremendous impact on the quality of life of CF patients (6,7). To enhance accurate identification of CF pathogens, several PCR assays have been developed recently (28,30,32-35). We sought to design similar PCR tests to allow the identification of R. pickettii and R. mannitolilytica based on species-level signature sequences in the 16S rRNA gene. By comparing available R. pickettii and R. mannitolilytica 16S rRNA gene sequences with sequences from other Ralstonia species and representatives of the phylogenetically closely related genera Burkholderia and Pandoraea, we identified several regions that showed sufficient diversity to allow the design of primer pairs Rp-F1/Rp-R1 and Rm-F1/Rm-R1, permitting the sensitive and specific identification of R. pickettii and R. mannitolilytica, respectively (Table).
The results of our study indicate that a number of Ralstonia species can be isolated from sputum cultures of CF patients. The correct identification of these species presents a challenge for diagnostic microbiology laboratories. Our study supports the use of genotypic methods to augment routine phenotypic evaluation. The combined use of the two PCR assays described will allow the identification of most Ralstonia species encountered in sputum cultures of CF patients. Most importantly Adv. 1. most importantly - above and beyond all other consideration; "above all, you must be independent"
above all, most especially , the use of these assays will substantially reduce the misidentification of R. pickettii and R. mannitolilytica as B. cepacia complex. These tests will be a valuable adjunct in the evaluation of CF sputum culture isolates and will allow more precise study of the prevalence and natural history of human infection by these emerging pathogens.
Table. Sensitivity and specificity of polymerase chain reaction (PCR) assays for the identification of Ralstonia mannitolilytica and R. pickettii Primer pair and species tested Sensitivity (%) Specificity (%) Rp-F1/Rp-R1 R. pickettii (n=27) 89 99 All others (n=125) Rm-F1/Rm-R1 R. mannitolilytica 100 99 (n=34) All others (n=118) No. of strains Primer pair and species tested Positive Negative Rp-F1/Rp-R1 R. pickettii (n=27) 24 3 (a) All others (n=125) 1(b) 124 Rm-F1/Rm-R1 R.mannitolilytica 34 0 (n=34) All others (n=118) 1 (c) 117 (a) Of the three R. pickettii isolates that gave a false-negative reaction in this PCR test, two were identified as R. pickettii by the referring laboratory, while one was received unidentified. (b) One unidentified Ralstonia sp. isolate cross-reacted with this primer pair. (c) One R. pickettii isolate cross-reacted with this primer pair.
We thank T. Spilker and A. Martin for excellent technical assistance.
This work was supported by a grant from the Cystic Fibrosis Foundation The Cystic Fibrosis Foundation (CFF) is a non-profit organization in the United States established to provide the means to cure and control cystic fibrosis. The Foundation provides information about cystic fibrosis (CF) and finances CF research that aims to improve the (United States United States, officially United States of America, republic (2005 est. pop. 295,734,000), 3,539,227 sq mi (9,166,598 sq km), North America. The United States is the world's third largest country in population and the fourth largest country in area. ) (to JJL JJL Jeunesse Juive Laique (Brussels, Belgium youth movement)
JJL Josephson Junction Logic ). TC is supported by the Caroll Haas Research Fund in Cystic Fibrosis.
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Dr. Coenye is a postodoctoral research fellow in the Department of Pediatrics and Communicable Diseases at the University of Michigan. His major research interests are the identification, biodiversity, and molecular epidemiology molecular epidemiology Molecular medicine An evolving field that combines the tools of standard epidemiology–case studies, questionnaires and monitoring of exposure to external factors with the tools of molecular biology–eg, restriction endonucleases, of bacteria associated with pulmonary infections in cystic fibrosis patients.
Tom Coenye, * Peter Vandamme, ([dagger]) and John J. LiPuma *
* University of Michigan Medical School, Ann Arbor, Michigan
“Ann Arbor” redirects here. For other uses, see Ann Arbor (disambiguation).
Ann Arbor is a city in the U.S. state of Michigan and the county seat of Washtenaw County. , USA; and ([dagger]) Ghent University, Ghent, Belgium
Address for correspondence: Tom Coenye, Department of Pediatrics and Communicable Diseases, 8301 MSRB MSRB
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Noorhamdani (Member): Infection by Ralstonia species in cystic fibrosis patients: identification of R. pickettii and R. mannitolilytica by polymerase chain reaction. (Research). 5/22/2010 8:47 AM
R solanacearums are plant pathogens. Why can R solanacearums infect human, especiallay humans suffer cystic fibrosis? How mechanism of pathogenesis R solanacearum infect human? Thak you for your attension. Noorhamdani.