Genetic Dissection of Antibiotic Adjuvant Activity.

Small molecule adjuvants that enhance the activity of established antibiotics represent promising agents in the battle against antibiotic resistance. Adjuvants generally act by inhibiting antibiotic resistance processes, and specifying the process acted on is a critical step in defining an adjuvant's mechanism of action. This step is typically carried out biochemically by identifying molecules that bind adjuvants and then inferring their roles in resistance. Here, we present a complementary genetic strategy based on identifying mutations that both sensitize cells to antibiotic and make them "adjuvant blind." We tested the approach in Acinetobacter baumannii AB5075 using two adjuvants: a well-characterized ß-lactamase inhibitor (avibactam) and a compound enhancing outer membrane permeability (aryl 2-aminoimidazole AI-1). The avibactam studies showed that the adjuvant potentiated one ß-lactam (ceftazidime) through action on a single ß-lactamase (GES-14) and a second (meropenem) by targeting two different enzymes (GES-14 and OXA-23). Mutations impairing disulfide bond formation (DsbAB) also reduced potentiation, possibly by impairing ß-lactamase folding. Mutations reducing AI-1 potentiation of canonical Gram-positive antibiotics (vancomycin and clarithromycin) blocked lipooligosaccharide (LOS/LPS) synthesis or its acyl modification. The results indicate that LOS-mediated outer membrane impermeability is targeted by the adjuvant and show the importance of acylation in the resistance. As part of the study, we employed Acinetobacter baylyi as a model to verify the generality of the A. baumannii results and identified the principal resistance genes for ceftazidime, meropenem, vancomycin, and clarithromycin in A. baumannii AB5075. Overall, the work provides a foundation for analyzing adjuvant action using a comprehensive genetic approach. IMPORTANCE One strategy to confront the antibiotic resistance crisis is through the development of adjuvant compounds that increase the efficacy of established drugs. A key step in the development of a natural product adjuvant as a drug is identifying the resistance process it undermines to enhance antibiotic activity. Previous procedures designed to accomplish this have relied on biochemical identification of cell components that bind adjuvant. Here, we present a complementary strategy based on identifying mutations that eliminate adjuvant activity.

Use of exogenous erythropoietin in critically ill patients.

OBJECTIVE: Review the literature regarding the use of recombinant human erythropoietin (rHuEPO) to prevent red blood cell (RBC) transfusion in critically ill patients. DATA SOURCES: A computerized search of MEDLINE and EMBASE from 1966 through June 2003 was conducted using the terms erythropoietin, anemia, hemoglobin, critical care, intensive care, surgery, trauma, burn, and transfusion. References of selected articles were reviewed. A manual search of critical care, surgery, trauma, burn, hematology, and pharmacy journals was conducted to identify relevant abstracts. RESULTS: Six randomized studies have evaluated exogenous administration of erythropoietin to prevent RBC transfusions in critically ill patients. Studies vary with respect to rHuEPO dosage regimens, dose of concurrently administered iron, patient characteristics, and transfusion thresholds. Administration of rHuEPO rapidly produces erythropoiesis to reduce the need for RBC transfusions. The largest study conducted to date used weekly rHuEPO administration and found a modest decrease in transfusion requirements although the time to first transfusion was delayed. Reduced intensive care unit (ICU) length of stay (LOS) was shown in only one study of surgical/trauma patients. Reduced LOS after ICU discharge was found in another study of severely ill patients (APACHE II score >22). Other clinical outcomes were not altered by rHuEPO use. No adverse events were associated with rHuEPO use although studies were not designed to evaluate safety. CONCLUSIONS: rHuEPO reduces the need for transfusions. A cost-effectiveness analysis of rHuEPO for this indication is needed. Defining an optimal dosage regimen, identifying patients most likely to respond to rHuEPO, and determining risk factors for ICU associated anaemia would provide information for appropriate rHuEPO utilization.

Songbird genomics: analysis of 45 kb upstream of a polymorphic Mhc class II gene in red-winged blackbirds (Agelaius phoeniceus).

Here we present the sequence of a 45 kb cosmid containing a previously characterized poly-morphic Mhc class II B gene (Agph-DAB1) from the red-winged blackbird (Agelaius phoeniceus). We compared it with a previously sequenced cosmid from this species, revealing two regions of 7.5 kb and 13.0 kb that averaged greater than 97% similarity to each another, indicating a very recent shared duplication. We found 12 retroelements, including two chicken repeat 1 (CR1) elements, constituting 6.4% of the sequence and indicating a lower frequency of retroelements than that found in mammalian genomic DNA. Agph-DAB3, a new class II B gene discovered in the cosmid, showed a low rate of polymorphism and may be functional. In addition, we found a Mhc class II B gene fragment and three genes likely to be functional (encoding activin receptor type II, a zinc finger, and a putative gamma-filamin). Phylogenetic analysis of exon 2 alleles of all three known blackbird Mhc genes indicated strong clustering of alleles by locus, implying that large amounts of interlocus gene conversion have not occurred since these genes have been diverging. Despite this, interspecific comparisons indicate that all three blackbird Mhc genes diverged from one another less than 35 million years ago and are subject to concerted evolution in the long term. Comparison of blackbird and chicken Mhc promoter regions revealed songbird promoter elements for the first time. The high gene density of this cosmid confirms similar findings for the chicken Mhc, but the segment duplications and diversity of retroelements resembles mammalian sequences.

A 39-kb sequence around a blackbird Mhc class II gene: ghost of selection past and songbird genome architecture.

To gain an understanding of the evolution and genomic context of avian major histocompatibility complex (Mhc) genes, we sequenced a 38.8-kb Mhc-bearing cosmid insert from a red-winged blackbird (Agelaius phoeniceus). The DNA sequence, the longest yet retrieved from a bird other than a chicken, provides a detailed view of the process of gene duplication, divergence, and degeneration ("birth and death") in the avian Mhc, as well as a glimpse into major noncoding features of a songbird genome. The peptide-binding region (PBR) of the single Mhc class II B gene in this region, Agph-DAB2, is almost devoid of polymorphism, and a still-segregating single-base-pair deletion and other features suggest that it is nonfunctional. Agph-DAB2 is estimated to have diverged about 40 MYA from a previously characterized and highly polymorphic blackbird Mhc gene, Aph-DAB1, and is therefore younger than most mammalian Mhc paralogs and arose relatively late in avian evolution. Despite its nonfunctionality, Agph-DAB2 shows very high levels of nonsynonymous divergence from Agph-DAB1 and from reconstructed ancestral sequences in antigen-binding PBR codons-a strong indication of a period of adaptive divergence preceding loss of function. We also found that the region sequenced contains very few other unambiguous genes, a partial Mhc- class II gene fragment, and a paucity of simple-sequence and other repeats. Thus, this sequence exhibits some of the genomic streamlining expected for avian as compared with mammalian genomes, but is not as densely packed with functional genes as is the chicken Mhc.

MHC class II pseudogene and genomic signature of a 32-kb cosmid in the house finch (Carpodacus mexicanus).

Large-scale sequencing studies in vertebrates have thus far focused primarily on the genomes of a few model organisms. Birds are of interest to genomics because of their much smaller and highly streamlined genomes compared to mammals. However, large-scale genetic work has been confined almost exclusively to the chicken; we know little about general aspects of genomes in nongame birds. This study examines the organization of a genomic region containing an Mhc class II B gene in a representative of another important lineage of the avian tree, the songbirds (Passeriformes). We used a shotgun sequencing approach to determine the sequence of a 32-kb cosmid insert containing a strongly hybridizing Mhc fragment from house finches (Carpodacus mexicanus). There were a total of three genes found on the cosmid clone, about the gene density expected for the mammalian Mhc: a class II Mhc beta-chain gene (Came-DAB1), a serine-threonine kinase, and a zinc finger motif. Frameshift mutations in both the second and third exons of Came-DAB1 and the unalignability of the gene after the third exon suggest that it is a nonfunctional pseudogene. In addition, the identifiable introns of Came-DAB1 are more than twice as large as those of chickens. Nucleotide diversity in the peptide-binding region of Came-DAB1 (Pi = 0.03) was much lower than polymorphic chicken and other functional Mhc genes but higher than the expected diversity for a neutral locus in birds, perhaps because of hitchhiking on a selected Mhc locus close by. The serine-threonine kinase gene is likely functional, whereas the zinc finger motif is likely nonfunctional. A paucity of long simple-sequence repeats and retroelements is consistent with emerging rules of chicken genomics, and a pictorial analysis of the "genomic signature" of this sequence, the first of its kind for birds, bears strong similarity to mammalian signatures, suggesting common higher-order structures in these homeothermic genomes. The house finch sequence is among a very few of its kind from nonmodel vertebrates and provides insight into the evolution of the avian Mhc and of avian genomes generally.

Toward an evolutionary genomics of the avian Mhc.

We review recent developments in the ongoing study of the evolution of the Mhc gene family in birds, with emphasis on class II B genes and results from songbirds obtained in our laboratory. Southern blots suggest a surprising diversity in Mhc class II gene number among various songbird species (Passeriformes). We have sequenced approximately 30 kb contigs from Mhc bearing cosmid clones from two species, red-winged blackbirds (Agelaius phoeniceus) and house finches (Carpodacus mexicanus), whose demography, lifetime reproductive success, epizootics, parasitology and mate choice are among the best studied for natural populations of birds. Of three genes cloned from these species, only one appears strongly polymorphic, and one (from the house finch) is likely a pseudogene. All are similar in structure to those in chickens, albeit with introns intermediate in length between chickens and mammals. Phylogenetic analysis of available class II B peptide-binding region exons suggests that the overwhelming long-term force operating on avian genes sampled thus far has been post-speciation gene duplication and/or concerted evolution. These and other results suggest that the evolution of class II B genes in birds conforms to a mixture of several models of multigene family evolution proposed for the mammalian Mhc, incorporating ongoing homogenization, duplication and pseudogene formation. Large-scale sequencing studies in these and other species, though still in their infancy, will prove invaluable for studying the comparative structures of avian Mhcs, as well as patterns of selection, mutation and linkage disequilibrium at several scales.

Genomics and polymorphism of Agph-DAB1, an Mhc class II B gene in red-winged blackbirds (Agelaius phoeniceus).

To further our understanding of the evolution of avian Mhc genes at the genomic level, we screened a cosmid library made from a red-winged blackbird (Agelaius phoeniceus) with a blackbird cDNA probe and subcloned from one of the Mhc-containing cosmids a gene which we designate Agph-DAB1. The structure of the gene is similar to that found for chicken class II B genes, except that the introns are surprisingly large, ranging from 98 to over 600 bp, making this the longest avian class II B gene to date. Using primers targeted toward the introns flanking the peptide-binding region (PBR), we amplified the entirety of the second exon and determined nucleotide sequences of 41 PCR products from eight individual blackbirds. The 10 sequence types found, among which were two probable pseudogene sequences, exhibit the classic hallmarks for evolution of PBRs, namely, an excess of nonsynonymous over synonymous substitutions and evidence of gene conversion events in polymorphic subdomains. Despite these patterns and our use of intron primers, the distribution of sequences among individuals suggests that more than one locus was amplified in most individuals, and the bushlike tree of sequences provides little information as to locus-specific clusters. These results imply a complex history of gene conversion, recent duplication, or possibly, concerted evolution among multiple loci, although Agph-DAB1, the first genomic Mhc sequence from a bird other than chicken, provides important clues in the quest for locus-specific Mhc primers in birds.

Transportation of radioactive material in Georgia.

A 3-yr study of radioactive materials transportation examined the magnitude of radioactive materials shipments in terms of numbers of packages and motor vehicle trips and types of materials; compliance with regulations for packaging, labelling, handling, external radiation exposure, and surface contamination; and dose to workers as measured with personnel dosimeters. Much of the information was obtained at the Atlanta airport and its vicinity, a package distribution center for the southeastern U.S., and at the Barnwell, S.C. radioactive waste burial site, the destination of most shipments of radioactive waste from or through Georgia. Approximately 12,000 packages in radioactive material categories I, II and III were handled in Georgia each year. Motor vehicles made approximately 3300 trips per year. Some instances of noncompliance were observed, but few of them had the potential for elevated radiation exposure of persons. Several incidents associated with radioactive material transport are reported, of which one may have resulted in slightly elevated exposures to persons. Among drivers and handlers who worked with radioactive material shipments, dosimeters showed that less than one-half of them received radiation doses above background levels. The highest doses were found for drivers who transported large numbers of 99Mo generators.