'Haptoglobin concentrations in preterm and term newborns'.

OBJECTIVE: To measure systemic haptoglobin (HPT) concentrations from birth in preterm (PT) and T newborns. To compare HPT in newborns without hemolysis or infection with values in bacteremic newborns. STUDY DESIGN: HPT was measured using enzyme-linked immunosorbent assay in 30 PT and 28 T newborns without hemolysis or infection at birth (cord blood), on days of life 2 to 4, and at 1 to 2 weeks of life. Concentrations were measured in eight additional newborns with bacteremia. Wilcoxon-Mann-Whitney test was used for comparisons. RESULT: HPT concentrations were consistently measurable from birth in PT and T neonates. Values were significantly greater in 2- to 4-day-old PT and T newborns than in newborns at birth (P<0.01). Bacteremic newborns had higher HPT concentrations than newborns without infection (P=0.033). CONCLUSION: HPT is detectable from birth in PT and T newborns. HPT concentrations increase in bacteremic newborns. HPT levels may have clinical utility in the evaluation of neonatal sepsis.

Identification and detection of Stenotrophomonas maltophilia by rRNA-directed PCR.

Stenotrophomonas maltophilia has recently emerged as an important nosocomial pathogen in immunocompromised patients, in transplant recipients, and in persons with cystic fibrosis (CF). While this organism is nonpathogenic in healthy individuals, it is increasingly associated with morbidity and mortality in susceptible populations. Recent studies have indicated that for approximately 10% of CF patients with moderate lung disease, S. maltophilia can be cultured from respiratory tract secretions. Identification of S. maltophilia can be problematic, and analysis of isolates from the Burkholderia cepacia Research Laboratory and Repository showed that several isolates presumptively identified as B. cepacia by clinical microbiology laboratories were in fact S. maltophilia. To overcome the problems associated with definitive identification, we developed species-specific PCR (SS-PCR) primers, designated SM1 and SM4, directed to the 23S rRNA gene, and tested their utility to accurately identify S. maltophilia directly from sputum. The SS-PCR was developed and tested against a panel of 112 S. maltophilia isolates collected from diverse geographic locations. To test for specificity, 43 isolates from 17 different species were analyzed. PCR with the SM1-SM4 primer pair and isolated genomic DNA as a template resulted in amplification of a band from all S. maltophilia isolates and was uniformly negative for all other species tested, yielding a sensitivity and a specificity of 100% for the SS-PCR. The utility of the SS-PCR to directly identify S. maltophilia in sputum was examined. Thirteen expectorated sputum samples from CF patients were analyzed by SS-PCR. Three samples were PCR positive, in complete concordance with the conventional laboratory culture. Thus, we have developed an SS-PCR protocol that can rapidly and accurately identify S. maltophilia isolates and which can be used for the direct detection of this organism in CF patient sputum.

Caring for Oklahoma's children: the Department of Pediatrics and Children's Hospital of Oklahoma.

The University of Oklahoma Department of Pediatrics was founded in 1930. Its history has mirrored the cyclic history of the state's economic development. The Department has maintained its commitment as a central resource for specialty care for sick children, training future physicians, and creating new information to improve children's health. The Department is currently in a stage of growth based on support from the College of Medicine and the community. Because the histories of the Department and the Children's Hospital of Oklahoma are intertwined, this article gives an overview of each.

Identification of members of the Burkholderia cepacia complex by species-specific PCR.

Definitive identification of the species in the Burkholderia cepacia complex by routine clinical microbiology methods is difficult. Phenotypic tests to identify B. multivorans and B. vietnamiensis have been established; more recent work indicates B. stabilis may also be identified by growth characteristics and biochemical tests. However, attempts to identify genomovars I and III have, thus far, proved unsuccessful. Previously, we demonstrated the utility of two primer pairs, directed to the rRNA operon, to specifically identify the B. cepacia complex in a PCR. One of these primer pairs, G1-G2, only amplified a DNA fragment from genomovars I and III and B. stabilis in a PCR with genomic DNA isolated from prototypical strains representing the five genomovars. Sequence analysis of the rRNA operon for all the genomovars indicated that this primer pair targeted a region shared by these isolates. Further analysis revealed a region of heterogeneity between genomovar III and B. stabilis internal to the amplified product of G1-G2. Primers designed to target this region were tested with prototypical strains following an initial amplification with the G1-G2 primer pair. New primers specific for the prototypical genomovar III and B. stabilis were designated SPR3 and SPR4, respectively. Analysis of 93 isolates representing 18 genomovar I, 13 B. multivorans, 36 genomovar III, 11 B. stabilis, and 15 B. vietnamiensis isolates was performed. DNA from all isolates of genomovars I and III and B. stabilis was amplified by G1-G2. Genomovar III isolates yielded a product with SPR3/G1 while B. stabilis amplified with SPR4-G1. Genomovar I isolates were amplified by either SPR3-G1 or SPR4-G1, but not both. B. multivorans yielded a product with SPR3-G1 but not G1-G2, and B. vietnamiensis isolates were negative in all PCRs. Thus using an algorithm with G1-G2, SPR3-G1, and SPR4-G1 primers in a PCR analysis, genomovar III isolates can be separated from B. stabilis and the identity of B. multivorans and B. vietnamiensis can be confirmed.

Antibacterial agents in pediatrics.

From low birth weight infants to adolescents, physiologic and developmental differences underlie the marked differences in pharmacokinetics and pharmacodynamics of antibacterial agents. Certain diseases, such as cystic fibrosis, also can alter these parameters. This article describes the principles of pharmacokinetics and pharmacodynamics that are unique to children and that characterize the clinical application of selected antibacterial agents to infectious diseases in children.

Species-specific PCR as a tool for the identification of Burkholderia gladioli.

Burkholderia gladioli colonizes the respiratory tracts of patients with cystic fibrosis and chronic granulomatous disease. However, due to the high degree of phenotypic similarity between this species and closely related species in the Burkholderia cepacia complex, accurate identification is difficult. Incorrect identification of these species may have serious repercussions for the management of patients with cystic fibrosis. To develop an accurate procedure for the identification of B. gladioli, a molecular method to discriminate between this species and other species commonly isolated from the sputa of patients with cystic fibrosis was investigated. The 23S ribosomal DNA was cloned from several clinical isolates of B. gladioli, and the nucleotide sequence was determined. Computer-assisted sequence comparisons indicated four regions of the 23S rRNA specific for this species; these regions were used to design three primer pairs for species-specific PCR. Two of the primer pairs showed 100% sensitivity and specificity for B. gladioli when tested against a panel of 47 isolates comprising 19 B. gladioli isolates and 28 isolates of 16 other bacterial species. One of the primer pairs was further assessed for species specificity by using a panel of 102 isolates obtained from the Burkholderia cepacia Research Laboratory and Repository. The species-specific PCR was positive for 70 of 74 isolates of B. gladioli and was negative for all other bacterial species examined. Overall, this primer pair displayed a sensitivity and specificity of 96% (89 of 93) and 100%, respectively. These data demonstrate the potential of species-specific PCR for the identification of B. gladioli.

Development of rRNA-based PCR assays for identification of Burkholderia cepacia complex isolates recovered from cystic fibrosis patients.

PCR assays targeting rRNA genes were developed to identify species (genomovars) within the Burkholderia cepacia complex. Each assay was tested with 177 bacterial isolates that also underwent taxonomic analysis by whole-cell protein profile. These isolates were from clinical and environmental sources and included 107 B. cepacia complex strains, 23 Burkholderia gladioli strains, 20 Ralstonia pickettii strains, 10 Pseudomonas aeruginosa strains, 8 Stenotrophomonas maltophilia strains, and 9 isolates belonging to nine other species. The sensitivity and specificity of the 16S rRNA-based assay for Burkholderia multivorans (genomovar II) were 100 and 99%, respectively; for Burkholderia vietnamiensis (genomovar V), sensitivity and specificity were 87 and 92%, respectively. An assay based on 16S and 23S rRNA gene analysis of B. cepacia ATCC 25416 (genomovar I) was useful in identifying genomovars I, III, and IV as a group (sensitivity, 100%, and specificity, 99%). Another assay, designed to be specific at the genus level, identified all but one of the Burkholderia and Ralstonia isolates tested (sensitivity, 99%, and specificity, 96%). The combined use of these assays offers a significant improvement over previously published PCR assays for B. cepacia.

Role of CCAA nucleotide repeats in regulation of hemoglobin and hemoglobin-haptoglobin binding protein genes of Haemophilus influenzae.

Haemophilus influenzae utilizes hemoglobin and hemoglobin-haptoglobin as heme sources. The H. influenzae hemoglobin- and hemoglobin-haptoglobin binding protein genes, hgpA, hgpB, and hgpC, contain lengths of tetrameric CCAA repeats. Using an hgpA-lacZ translational gene fusion, we demonstrate phase-variable expression of lacZ associated with alteration in the length of the CCAA repeat region.

Distribution of a family of Haemophilus influenzae genes containing CCAA nucleotide repeating units.

A family of genes containing lengths of CCAA nucleotide repeating units directly following the sequence encoding the leader peptide has been identified in Haemophilus influenzae. The length of the CCAA repeats ranges from 6 to 43 and all of the identified genes encode proteins or predicted proteins with a significant homology to bacterial iron- or heme-related outer membrane proteins. We have previously shown that two of these gene products, HgpA and HgpB, bind hemoglobin and the hemoglobin-haptoglobin complex. Studies were performed to define the species distribution of the five identified genes and the CCAA repeats. We show that both the CCAA motif and the structural genes for hemoglobin and hemoglobin-haptoglobin binding are widely distributed among H. influenzae strains.

Effect of multiple mutations in the hemoglobin- and hemoglobin-haptoglobin-binding proteins, HgpA, HgpB, and HgpC, of Haemophilus influenzae type b.

Haemophilus influenzae requires heme for growth and can utilize hemoglobin and hemoglobin-haptoglobin as heme sources. We previously identified two hemoglobin- and hemoglobin-haptoglobin-binding proteins, HgpA and HgpB, in H. influenzae HI689. Insertional mutation of hgpA and hgpB, either singly or together, did not abrogate the ability to utilize or bind either hemoglobin or the hemoglobin-haptoglobin complex. A hemoglobin affinity purification method was used to isolate a protein of approximately 120 kDa from the hgpA hgpB double mutant. We have cloned and sequenced the gene encoding this third hemoglobin/hemoglobin-haptoglobin binding protein and designate it hgpC. Insertional mutation of hgpC did not affect the ability of the strain to utilize either hemoglobin or hemoglobin-haptoglobin. An hgpA hgpB hgpC triple mutant constructed by insertional mutagenesis showed a reduced ability to use the hemoglobin-haptoglobin complex but was unaltered in the ability to use hemoglobin. A second class of mutants was constructed in which the entire structural gene of each of the three proteins was deleted. The hgpA hgpB hgpC complete-deletion triple mutant was unable to utilize the hemoglobin-haptoglobin complex and showed a reduced ability to use hemoglobin. We have identified three hemoglobin/hemoglobin-haptoglobin-binding proteins in Haemophilus influenzae. Any one of the three proteins is sufficient to support growth with hemoglobin-haptoglobin as the heme source, and expression of at least one of the three is essential for hemoglobin-haptoglobin utilization. Although the three proteins play a role in hemoglobin utilization, an additional hemoglobin acquisition mechanism(s) exists.

hgpB, a gene encoding a second Haemophilus influenzae hemoglobin- and hemoglobin-haptoglobin-binding protein.

Haemophilus influenzae requires heme for growth and can utilize both hemoglobin and hemoglobin-haptoglobin as heme sources. We previously identified a hemoglobin- and hemoglobin-haptoglobin-binding protein, HgpA, in H. influenzae HI689. Mutation of hgpA did not affect binding or utilization of either heme source. The hgpA mutant exhibited loss of a 120-kDa protein and increased expression of a 115-kDa protein. These data suggested that at least one other gene product is involved in binding of these heme sources by H. influenzae. A 3.2-kbp PCR product derived from HI689 was cloned. The nucleotide sequence indicated a separate, distinct gene with high homology to hgpA, which would encode a 115-kDa protein. Primers were designed for directional cloning of the structural gene in the correct reading frame. Sonicates of induced Escherichia coli harboring the cloned open reading frame bound both hemoglobin and hemoglobin-haptoglobin. An insertion/deletion mutant of H. influenzae at the newly identified locus, designated hgpB, was constructed. The 115-kDa protein was not detected in the mutant after affinity purification using biotinylated hemoglobin. An hgpA hgpB double-mutant strain exhibited a reduced ability to utilize hemoglobin-haptoglobin, although it was unaltered in the ability to utilize hemoglobin. Affinity isolation of hemoglobin-binding proteins from the double mutant resulted in isolation of an approximately 120-kDa protein. Internal peptide sequencing revealed this protein to be a third distinct protein, highly homologous to HgpA and HgpB. In summary a second hemoglobin- and hemoglobin-haptoglobin-binding protein of H. influenzae has been identified and characterized, and the presence of an additional protein of similar function has been revealed.

Comparison of culture and PCR for detection of Burkholderia cepacia in sputum samples of patients with cystic fibrosis.

We investigated the utility of PCR to detect Burkholderia cepacia directly in sputum samples at two cystic fibrosis (CF) centers serving children and adults. Following liquefaction of the sputa by using N-acetyl-L-cysteine, DNA was isolated and analyzed by PCRs with three different primer pairs directed toward bacterial rRNA loci. Two primer pairs were putatively specific for B. cepacia. The other pair, which universally amplifies a band from all bacteria, served as a control. Sputum samples were obtained from 219 patients and analyzed independently by culture and by PCR to detect B. cepacia. The analyses were performed blinded with respect to each other. The results of the PCR with sputa demonstrated that the primers directed to the 16S loci demonstrated approximately 95% concordance with culture results and were more specific than those amplifying the 16S to 23S spacer region. In addition, the 16S primer pair putatively identified B. cepacia in seven patients whose sputa were culture negative at this time. Of these culture-negative patients, five had sputum samples that were culture positive for B. cepacia either prior or subsequent to this study. The results of this study indicate the utility of PCR as a diagnostic method for the rapid identification of B. cepacia in sputum samples of CF patients. We anticipate that improvements in our taxonomic understanding may allow the design of more specific primers for detection of each species of the B. cepacia complex in sputum samples.

Construction of antibiotic resistance cassettes with multiple paired restriction sites for insertional mutagenesis of Haemophilus influenzae.

Insertional mutagenesis of cloned genes coupled with site specific recombination into the genome of the parent organism is an ideal method for characterizing gene function. In this paper we describe the production and utility of two antibiotic resistance cassettes for use in Haemophilus influenzae. The mutagenic elements encode resistance to chloramphenicol or spectinomycin. Multiple paired restriction enzyme sites bound both cassettes. Use of these constructs to create mutants in H. influenzae demonstrated that the cassettes are readily incorporated into the genome in single copy and allow easy detection of mutant constructs. The insertions are stable following repeated in vitro passage. In addition, the elements are compatible with each other and allow the construction of multiple mutations within a single strain.

Transcription of genes encoding iron and heme acquisition proteins of Haemophilus influenzae during acute otitis media.

Unencapsulated Haemophilus influenzae is the second most common etiologic agent of otitis media in children. H. influenzae requires heme for aerobic growth in vitro and is able to utilize hemoglobin and complexes of heme-hemopexin, heme-albumin, and hemoglobin-haptoglobin and ferritransferrin as sources of iron and heme in vitro. Several of the acquisition mechanisms have been characterized and been shown to be heme repressible in vitro. However, little is known about the expression of heme and/or iron acquisition mechanisms during infections in the middle ear. This study was performed to determine if the genes encoding heme and iron acquisition proteins are transcribed during in vivo growth and to compare these findings with those for samples grown in vitro. Reverse transcriptase PCR (RT-PCR) was used to analyze total RNA fractions derived from in vitro- and in vivo-grown H. influenzae. Genes encoding the transferrin-binding proteins TbpA and TbpB, the 100-kDa hemopexin-binding protein HxuA, and the hemoglobin-binding protein HgpA were transcribed during otitis media. Twelve middle ear fluid samples were analyzed by blind RT-PCR to determine the transcriptional status of these genes in H. influenzae during otitis media. Five isolates had transcripts corresponding to tbpA, tbpB, and hxuA. The presence of hgpA transcripts was variable, depending on the presence of hgpA in the genome of the H. influenzae isolate. Samples without H. influenzae gene transcripts contained other etiologic agents commonly causing otitis media. These data demonstrate that H. influenzae iron and/or heme acquisition genes are transcribed during otitis media and suggest that the microenvironment during acute otitis media starves H. influenzae of heme.

An outbreak of Burkholderia cepacia lower respiratory tract infection associated with contaminated albuterol nebulization solution.

An outbreak of Burkholderia cepacia lower respiratory tract colonization and infection occurred in the adult intensive-care units in various geographic locations throughout our hospital. Forty-four patients became colonized or infected over an 11-month period, whereas B cepacia had been isolated from only 13 patients in the preceding 48 months. Environmental cultures revealed the source to be extrinsically contaminated albuterol nebulization solution. Polymerase chain reaction-ribotyping confirmed the genetic relatedness of the B cepacia patient isolates and the contaminated albuterol. After extensive infection control training for the respiratory therapy staff, including attention to nebulization technique, washing and drying the nebulizer cup, and good handwashing, there have not been any new cases.

Cloning of a DNA fragment encoding a heme-repressible hemoglobin-binding outer membrane protein from Haemophilus influenzae.

Haemophilus influenzae is able to use hemoglobin as a sole source of heme, and heme-repressible hemoglobin binding to the cell surface has been demonstrated. Using an affinity purification methodology, a hemoglobin-binding protein of approximately 120 kDa was isolated from H. influenzae type b strain HI689 grown in heme-restricted but not in heme-replete conditions. The isolated protein was subjected to N-terminal amino acid sequencing, and the derived amino acid sequence was used to design corresponding oligonucleotides. The oligonucleotides were used to probe a Southern blot of EcoRI-digested HI689 genomic DNA. A hybridizing band of approximately 4.2 kb was successfully cloned into pUC19. Using a 1.9-kb internal BglII fragment of the 4.2-kb clone as a probe, hybridization was seen in both typeable and nontypeable H. influenzae but not in other bacterial species tested. Following partial nucleotide sequencing of the 4.2-kb insert, a putative open reading frame was subcloned into an expression vector. The host Escherichia coli strain in which the cloned fragment was expressed bound biotinylated human hemoglobin, whereas binding of hemoglobin was not detected in E. coli with the vector alone. In conclusion, we hypothesize that the DNA fragment encoding an approximately 120-kDa heme-repressible hemoglobin-binding protein mediates one step in the acquisition of hemoglobin by H. influenzae in vivo.

Antibacterial agents in pediatrics.

The utility of various antibacterial agents for therapy of infectious diseases in children is determined by the unique pharmacokinetics and potential toxicity in children. The important age-related principle of pharmacokinetics is reviewed in the first section of this article; the second section focuses on specific therapeutic agents and their use in children. Particular emphasis is given to the use of new antibiotics in children, including the new oral cephalosporins and macrolides.

Detection of Pseudomonas (Burkholderia) cepacia using PCR.

Pseudomonas cepacia colonization of the lung is associated with increased morbidity and mortality for cystic fibrosis (CF) patients. The lack of a sensitive detection method for Pseudomonas cepacia in CF sputum has resulted in controversy regarding its epidemiology. We designed a PCR method to detect P. cepacia using P. cepacia 16 S rRNA sequences as the amplification target region. The PCR amplification with purified DNA as template yielded the expected 209-bp products from P. cepacia, but not from related Pseudomonas species of medical importance or other bacteria which have been reported to colonize CF patients. In serial dilution experiments as few as 10(2) P. cepacia CFU were detectable. When sputum samples from three CF patients chronically colonized with P. cepacia and P. aeruginosa were analyzed, P. cepacia was detected in all three specimens by PCR, but only in two when selective culture was performed. Our data support the potential role of PCR technology in the rapid, sensitive, and definitive detection of P. cepacia in CF sputum samples, even in the context of concomitant P. aeruginosa colonization.

Production and oxidation of indole by Haemophilus influenzae.

During growth in high concentrations of iron nitrate, H. influenzae produces compounds reactive in biochemical assays for hydroxamates. Mixing experiments established that nitrate was responsible for inducing these compounds. Analysis by 1H and 13C NMR and high resolution mass spectrometry identified the active species as 2,2-bis(3'-indolyl)indoxyl. Bacterial production of the latter compound has been previously observed only in Pseudomonas aureofaciens. A mutant defective in the production of 2,2-bis(3'-indolyl)indoxyl was constructed by marker insertion. The formation of indole and 2,2-bis (3'-indolyl)indoxyl was quantitated by reverse-phase high pressure liquid chromatography during growth in high concentrations of nitrate. The mutant produced high concentrations of indole, but only minimal amounts of 2,2-bis(3'-indolyl)indoxyl, and also proved to be defective in nitrate reduction. These data suggest that indole may function as an electron donor for nitrate reductase in H. influenzae.