Development of a method to produce hemoglobin in a bioreactor culture of Escherichia coli BL21(DE3) transformed with a plasmid containing Plesiomonas shigelloides heme transport genes and modified human hemoglobin genes.

We describe a method for production of recombinant human hemoglobin by Escherichia coli grown in a bioreactor. E. coli BL21(DE3) transformed with a plasmid containing hemoglobin genes and Plesiomonas shigelloides heme transport genes reached a cell dry weight of 83.64 g/liter and produced 11.92 g/liter of hemoglobin in clarified lysates.

Enhancement of recombinant hemoglobin production in Escherichia coli BL21(DE3) containing the Plesiomonas shigelloides heme transport system.

To produce recombinant hemoglobin in Escherichia coli, sufficient intracellular heme must be present, or the protein folds improperly and is degraded. In this study, coexpression of human hemoglobin genes and Plesiomonas shigelloides heme transport genes enhanced recombinant hemoglobin production in E. coli BL21(DE3) grown in medium containing heme.

Characterization of the Plesiomonas shigelloides genes encoding the heme iron utilization system.

Plesiomonas shigelloides is a gram-negative pathogen which can utilize heme as an iron source. In previous work, P. shigelloides genes which permitted heme iron utilization in a laboratory strain of Escherichia coli were isolated. In the present study, the cloned P. shigelloides sequences were found to encode ten potential heme utilization proteins: HugA, the putative heme receptor; TonB and ExbBD; HugB, the putative periplasmic binding protein; HugCD, the putative inner membrane permease; and the proteins HugW, HugX, and HugZ. Three of the genes, hugA, hugZ, and tonB, contain a Fur box in their putative promoters, indicating that the genes may be iron regulated. When the P. shigelloides genes were tested in E. coli K-12 or in a heme iron utilization mutant of P. shigelloides, hugA, the TonB system genes, and hugW, hugX, or hugZ were required for heme iron utilization. When the genes were tested in a hemA entB mutant of E. coli, hugWXZ were not required for utilization of heme as a porphyrin source, but their absence resulted in heme toxicity when the strains were grown in media containing heme as an iron source. hugA could replace the Vibrio cholerae hutA in a heme iron utilization assay, and V. cholerae hutA could complement a P. shigelloides heme utilization mutant, suggesting that HugA is the heme receptor. Our analyses of the TonB system of P. shigelloides indicated that it could function in tonB mutants of both E. coli and V. cholerae and that it was similar to the V. cholerae TonB1 system in the amino acid sequence of the proteins and in the ability of the system to function in high-salt medium.

Design and implementation of a controlled trial of pediatric endotracheal intubation in the out-of-hospital setting.

This article describes the design and implementation of the Pediatric Airway Management Project. The project was completed January 1, 1997, and evaluated the effectiveness of endotracheal intubation relative to bag-valve-mask ventilation in improving survival to hospital discharge and neurologic outcome in children, the effect of training on paramedic airway management skills and self-efficacy, the length of time the skills can be retained, and the costs of training and retraining. The main focus of project design was the implementation of a controlled trial comparing methods of airway management for acutely ill and injured pediatric patients in the out-of-hospital setting. To date, this project is the largest prospective, controlled, out-of-hospital study of the care of children ever reported. Barriers to implementation of a study of this size are described.

Survey of nationally registered emergency medical services providers: pediatric education.

STUDY OBJECTIVE: To survey emergency medical services (EMS) providers on a national level to determine and describe their perspective regarding their initial and continuing education (CE) needs in pediatrics. METHODS: A 10-question survey was developed, pilot-tested, and sent to EMS providers as a part of their National Registry of Emergency Medical Technicians reregistration materials. RESULTS: Surveys were completed by 18,218 EMS providers, a response rate of 67%. During a typical month, 60% of emergency medical technician-paramedics (EMT-Ps), 84% of EMT-intermediates (EMT-Is), and 87% of basic EMTs (EMT-Bs) care for 0 to 3 pediatric patients. CE was identified by all provider levels as the main source of their pediatric knowledge and skills. A state or national mandate for required CE in pediatrics was supported by 76% of surveyed providers. More than 70% of all providers responded they were comfortable to some degree with their own ability and their EMS system's ability when confronted with a critical pediatric call. Cost, availability, and travel distance were identified by all levels as the primary barriers to obtaining pediatric CE. All levels identified infants as the age of greatest concern if the provider was called to manage a critical case. CONCLUSION: Surveyed practicing nationally registered EMS providers have infrequent contact with pediatric patients and have acquired most of their pediatric knowledge and skills from CE. In general, these providers are comfortable with their personal and their system's ability to care for children, but clearly support the need for required pediatric CE and identify the birth to 3-year age range as the priority for an educational focus. Cost, travel distance, and availability of pediatric CE are barriers that should be considered if pediatric CE is to be required of EMS providers.

Effect of out-of-hospital pediatric endotracheal intubation on survival and neurological outcome: a controlled clinical trial.

CONTEXT: Endotracheal intubation (ETI) is widely used for airway management of children in the out-of-hospital setting, despite a lack of controlled trials demonstrating a positive effect on survival or neurological outcome. OBJECTIVE: To compare the survival and neurological outcomes of pediatric patients treated with bag-valve-mask ventilation (BVM) with those of patients treated with BVM followed by ETI. DESIGN: Controlled clinical trial, in which patients were assigned to interventions by calendar day from March 15, 1994, through January 1, 1997. SETTING: Two large, urban, rapid-transport emergency medical services (EMS) systems. PARTICIPANTS: A total of 830 consecutive patients aged 12 years or younger or estimated to weigh less than 40 kg who required airway management; 820 were available for follow-up. INTERVENTIONS: Patients were assigned to receive either BVM (odd days; n = 410) or BVM followed by ETI (even days; n = 420). MAIN OUTCOME MEASURES: Survival to hospital discharge and neurological status at discharge from an acute care hospital compared by treatment group. RESULTS: There was no significant difference in survival between the BVM group (123/404 [30%]) and the ETI group (110/416 [26%]) (odds ratio [OR], 0.82; 95% confidence interval [CI], 0.61-1.11) or in the rate of achieving a good neurological outcome (BVM, 92/404 [23%] vs ETI, 85/416 [20%]) (OR, 0.87; 95% CI, 0.62-1.22). CONCLUSION: These results indicate that the addition of out-of-hospital ETI to a paramedic scope of practice that already includes BVM did not improve survival or neurological outcome of pediatric patients treated in an urban EMS system.

Comparison of the heme iron utilization systems of pathogenic Vibrios.

Vibrio alginolyticus, Vibrio fluvialis, and Vibrio parahaemolyticus utilized heme and hemoglobin as iron sources and contained chromosomal DNA similar to several Vibrio cholerae heme iron utilization genes. A V. parahaemolyticus gene that performed the function of V. cholerae hutA was isolated. A portion of the tonB1 locus of V. parahaemolyticus was sequenced and found to encode proteins similar in amino acid sequence to V. cholerae HutW, TonB1, and ExbB1. A recombinant plasmid containing the V. cholerae tonB1 and exbB1D1 genes complemented a V. alginolyticus heme utilization mutant. These data suggest that the heme iron utilization systems of the pathogenic vibrios tested, particularly V. parahaemolyticus and V. alginolyticus, are similar at the DNA level, the functional level, and, in the case of V. parahaemolyticus, the amino acid sequence or protein level to that of V. cholerae.

Vibrio cholerae iron transport: haem transport genes are linked to one of two sets of tonB, exbB, exbD genes.

Vibrio cholerae was found to have two sets of genes encoding TonB, ExbB and ExbD proteins. The first set (tonB1, exbB1, exbD1) was obtained by complementation of a V. cholerae tonB mutant. In the mutant, a plasmid containing these genes permitted transport via the known V. cholerae high-affinity iron transport systems, including uptake of haem, vibriobactin and ferrichrome. When chromosomal mutations in exbB1 or exbD1 were introduced into a wild-type V. cholerae background, no defect in iron transport was noted, indicating the existence of additional genes that can complement the defect in the wild-type background. Another region of the V. cholerae chromosome was cloned that encoded a second functional TonB/Exb system (tonB2, exbB2, exbD2). A chromosomal mutation in exbB2 also failed to exhibit a defect in iron transport, but a V. cholerae strain that had chromosomal mutations in both the exbB1 and exbB2 genes displayed a mutant phenotype similar to that of an Escherichia coli tonB mutant. The genes encoding TonB1, ExbB1, ExbD1 were part of an operon that included three haem transport genes (hutBCD), and all six genes appeared to be expressed from a single Fur-regulated promoter upstream of tonB1. A plasmid containing all six genes permitted utilization of haem by an E. coli strain expressing the V. cholerae haem receptor, HutA. Analysis of the hut genes indicated that hutBCD, which are predicted to encode a periplasmic binding protein (HutB) and cytoplasmic membrane permease (HutC and HutD), were required to reconstitute the V. cholerae haem transport system in E. coli. In V. cholerae, the presence of hutBCD stimulated growth when haemin was the iron source, but these genes were not essential for haemin utilization in V. cholerae.

Implementation of the recommendations of the Institute of Medicine report, Emergency medical services for children: registered nurses.

Registered nurses are the single largest group of health care providers in the United States. Their role in emergency medical services for children and approaches to implementation of the Institute of Medicine recommendations as they relate to nursing care are discussed.

Effect of transportation and acid neutralization on recovery of mycobacteria from processed specimens.

Because of the increasing incidence of tuberculosis, more rapid detection of mycobacteria has become an important issue. Realizing that not every clinical laboratory has a rapid detection system for growing mycobacteria, this study was conducted to examine the feasibility of submitting sediments of processed specimens to a reference laboratory for further testing in a radiometric system. Using N-acetyl-L-cysteine-NaOH solution, 247 respiratory specimens were processed at a diagnostic laboratory. Half of each sediment was cultured on conventional media. The remainder was kept at 4 degrees C for a period of up to 1 week before transportation to a reference laboratory for culture by BACTEC system. Both laboratories recovered 25 organisms: 15 as Mycobacterium tuberculosis (MT) and 10 as M avium complex (MAC). Additionally, mycobacteria (MT[3], MAC[6], M gordonae [4], and M fortuitum [1]) were recovered from 14 specimens by the diagnostic laboratory that were not grown by the reference laboratory. These results indicate a significant decrease in viability of mycobacteria after processing of the specimens. Acid neutralization of the digested respiratory sediments significantly improved the recovery rate of mycobacteria even after 2 days of delay in culture. This preliminary work suggests that more extensive studies will provide useful information to delineate approaches to submitting neutralized sediments for mycobacterial cultures.

Vibrio cholerae iron transport systems: roles of heme and siderophore iron transport in virulence and identification of a gene associated with multiple iron transport systems.

Vibrio cholerae iron transport mutants were tested for their ability to cause disease in an infant mouse model. The mice were challenged with either the wild-type strain, a vibriobactin synthesis mutant, a heme utilization mutant, or double mutants containing both the vibriobactin synthesis defect and the heme utilization defect. When mice were challenged with 10(7) bacteria, the ability of the double mutant to survive in the intestines was greatly reduced and that of the heme utilization mutant was slightly reduced compared with that of the wild type or the vibriobactin synthesis mutant. When the inoculum size was reduced 10-fold, all of the iron transport mutants failed to colonize the intestines and failed to cause diarrhea in the mice, whereas the wild-type strain was not cleared and elicited a diarrheal response. These data indicate that disruption of either the heme utilization or the vibriobactin uptake system reduces the ability of V. cholerae to cause disease. One of the heme utilization mutants, DHH1, was found to be defective also in utilization of vibriobactin and ferrichrome, mimicking the Escherichia coli TonB- phenotype. This mutant was the least virulent of the iron transport mutants tested. Transformation of DHH1 with the recombinant plasmid pHUT4 restored the abilities to use hemin, vibriobactin, and ferrichrome as iron sources, suggesting that pHUT4 encodes a gene(s) involved globally in the iron transport systems. Hybridization of Vibrio DNA with the V. cholerae heme utilization genes demonstrated the presence of DNA homologous to the genes encoding the outer membrane protein HutA and the inner membrane protein HutB in all the V. cholerae strains tested. The probe containing hutA, but not that containing hutB, also hybridized to DNA from Vibrio parahaemolyticus.

Characterization of the Vibrio cholerae outer membrane heme transport protein HutA: sequence of the gene, regulation of expression, and homology to the family of TonB-dependent proteins.

The regulation of hutA, the Vibrio cholerae gene encoding a 77-kDa iron-regulated outer membrane protein required for heme iron utilization, was characterized, and the DNA sequence of the gene was determined. A hutA::Tn5 lac fusion generated previously (D. P. Henderson and S. M. Payne, Mol. Microbiol. 7:461-469, 1993) was transformed into Fur- and Fur+ strains of Escherichia coli and V. cholerae. The results of beta-galactosidase assays on the transformed strains demonstrated that transcription of hutA is regulated by the Fur repressor protein in E. coli and at least partially regulated by Fur in V. cholerae. Analysis of the DNA sequence of hutA indicated that a sequence homologous to the E. coli consensus Fur box was present in the promoter region of hutA. The amino acid sequence of HutA is homologous to those of several TonB-dependent outer member proteins. However, when the V. cholerae heme utilization system, which requires one or more genes encoded by the recombinant plasmid pHUT10 in addition to hutA carried on a second vector, was transferred to a wild-type strain and an isogenic tonB mutant of E. coli, the tonB mutant could utilize heme iron as efficiently as the wild-type strain. These data indicate that the V. cholerae heme utilization system reconstituted in E. coli does not require a functional TonB protein. The tonB mutant transformed with the heme utilization plasmids could not utilize the siderophore ferrichrome as an iron source, indicating that none of the genes encoded on the heme utilization plasmids complements the tonB defect in E. coli. It is possible that a gene(s) encoded by the recombinant heme utilization plasmids encodes a protein serving a TonB-like function in V. cholerae. A region in the carboxy terminus of HutA is homologous to the horse hemoglobin gamma chain, and the amino acids involved in forming the heme pocket in the gamma chain are conserved in HutA. These data suggest that this region of HutA is involved in heme binding.

Cloning and characterization of the Vibrio cholerae genes encoding the utilization of iron from haemin and haemoglobin.

Vibrio cholerae can utilize haemin or haemoglobin as its sole source of iron. Four haem utilization mutants of a classical strain of V. cholerae were isolated. These mutations were complemented with pHUT1, a cosmid clone isolated from a library of wild-type CA401 DNA. Two independent Tn5 insertions into the cloned sequence disrupted function in all of the complemented mutants. Escherichia coli 1017 transformed with pHUT1 failed to utilize haemin as an iron source; a second plasmid containing a different cloned fragment of V. cholerae DNA (pHUT3) was required in addition to pHUT1 to reconstitute the system in E. coli. Minicell analysis and SDS-PAGE of protein fractions indicate that pHUT10 (a subclone of pHUT1) encodes a 26 kDa inner membrane protein, and pHUT3 encodes a 77 kDa outer membrane protein. Loss of either protein by Tn5 mutagenesis abolishes haem utilization in E. coli. An E. coli hemA mutant that cannot synthesize porphyrins was transformed with the recombinant plasmids to determine whether the plasmids encoded the ability to transport the porphyrin as well as the iron. The transformants grew aerobically in media containing haemin, whereas the parental strain was unable to grow under these conditions. This indicates that V. cholerae haem-iron utilization genes allow transport of the entire haem moiety into the cell.

Education in pediatric basic and advanced life support.

Curricula in pediatric resuscitation must be based on adult learning principles. The Pediatric Basic and Advanced Life Support Courses (PBLS, PALS) should use educational strategies fostering positive interactions between the instructor and learners and should take into consideration the learner's motivation for taking the course. Materials should be developed for a specific target audience; course design should be flexible to meet individual needs. The PBLS and PALS courses have different audiences. PBLS is targeted toward caretakers of children. This course will continue to emphasize primary injury prevention as the major strategy but also will give learners the knowledge and skills to perform CPR. To optimize retention of knowledge and skills, the course content must be simplified, and the sequencing of steps in basic life support must be modified. Health care providers caring for pediatric patients in acute care settings are the primary audience for the PALS course. This course already incorporates adult learning principles; only minor revisions are anticipated. Discussion of the trauma patient is limited in the PALS course; additional trauma education could be facilitated by the addition of a trauma module or by other educational courses. All aspects of the PBLS and PALS educational programs must be evaluated continually to determine whether learning objectives have been met and whether the teaching format is appropriate. The answers to evaluation questions will help determine the degree to which the American Heart Association is meeting its educational objectives and how to allocate resources for development and training.

Pediatric prehospital care in urban and rural areas.

There are limited data concerning pediatric prehospital care, although pediatric prehospital calls constitute 10% of emergency medical services activity. Data from 10,493 prehospital care reports in 11 counties of California (four emergency medical services systems in rural and urban areas) were collected and analyzed. Comparison of urban and rural data found few significant differences in parameters analyzed. Use of the emergency medical services system by pediatric patients increased with age, but 12.5% of all calls were for children younger than 2 years. Calls for medical problems were most common for patients younger than 5 years of age; trauma was a more common complaint in rural areas (64%, P = .0001). Frequency of vital sign assessment differed by region, as did hospital contact (P less than .0001). Complete assessment of young pediatric patients, with a full set of vital signs and neurologic assessment, was rarely performed. Advanced life support providers were often on the scene, but advanced life support treatments and procedures were infrequently used. This study suggests the need for additional data on which to base emergency medical services system design and some directions for education of prehospital care providers.