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Douglas R Call

Publications and source records attributed to Douglas R Call.

At least 19 recordsLinked to original sources
PubMed2007

Validation of mixed-genome microarrays as a method for genetic discrimination.

Comparative genomic hybridizations have been used to examine genetic relationships among bacteria. The microarrays used in these experiments may have open reading frames from one or more reference strains (whole-genome microarrays), or they may be composed of random DNA fragments from a large number of strains (mixed-genome microarrays [MGMs]). In this work both experimental and virtual arrays are analyzed to assess the validity of genetic inferences from these experiments with a focus on MGMs. Empirical data are analyzed from an Enterococcus MGM, while a virtual MGM is constructed in silico using sequenced genomes (Streptococcus). On average, a small MGM is capable of correctly deriving phylogenetic relationships between seven species of Enterococcus with accuracies of 100% (n=100 probes) and 95% (n=46 probes); more probes are required for intraspecific differentiation. Compared to multilocus sequence methods and whole-genome microarrays, MGMs provide additional discrimination between closely related strains and offer the possibility of identifying unique strain or lineage markers. Representational bias can have mixed effects. Microarrays composed of probes from a single genome can be used to derive phylogenetic relationships, although branch length can be exaggerated for the reference strain. We describe a case where disproportional representation of different strains used to construct an MGM can result in inaccurate phylogenetic inferences, and we illustrate an algorithm that is capable of correcting this type of bias. The bias correction algorithm automatically provides bootstrap confidence values and can provide multiple bias-corrected trees with high confidence values.

Algorithms↗
PubMed2006

Identification of specific gene sequences conserved in contemporary epidemic strains of Salmonella enterica.

Genetic elements specific to recent and contemporary epidemic strains of Salmonella enterica were identified using comparative genomic analysis. Two epidemic multidrug-resistant (MDR) strains, MDR Salmonella enterica serovar Typhimurium definitive phage type 104 (DT104) and cephalosporin-resistant MDR Salmonella enterica serovar Newport, and an epidemic pansusceptible strain, Salmonella serovar Typhimurium DT160, were subjected to Salmonella gene microarray and suppression subtractive hybridization analyses. Their genome contents were compared with those of coexisting sporadic strains matched by serotype, geographic and temporal distribution, and host species origin. These paired comparisons revealed that epidemic strains of S. enterica had specific genes and gene regions that were shared by isolates of the same subtype. Most of these gene sequences are related to mobile genetic elements, including phages, plasmids, and plasmid-like and transposable elements, and some genes may encode proteins conferring growth or survival advantages. The emergence of epidemic MDR strains may therefore be associated with the presence of fitness-associated genetic factors in addition to their antimicrobial resistance genes.

Animals↗
PubMed2006

Antimicrobial resistance in Salmonella enterica serovar Dublin isolates from beef and dairy sources.

Salmonella enterica serovar Dublin (S. Dublin) is a cattle-adapted Salmonella serovar, so if antimicrobial resistance in S. Dublin arises as a result of antimicrobial use this most likely occurs within the cattle reservoir without impact from antimicrobial use in humans. We tested the antimicrobial resistance of bovine-origin S. Dublin isolates from 1986 through 2004 using a standard disk diffusion method. High proportions of isolates throughout the time period were resistant to one or more antimicrobials, and a marked increase in resistance to ceftazidime occurred between 2000 and 2004. Dairy-origin isolates were more likely to be resistant to several antibiotics than were isolates from beef operations where exposure to antimicrobials is likely to be less frequent. Plasmid analysis of a subset of isolates also supported the hypothesis that antimicrobial resistance traits in the cattle-adapted serovar Dublin were acquired within the bovine host environment.

Animals↗
PubMed2006

Use of a nonmedicated dietary supplement correlates with increased prevalence of streptomycin-sulfa-tetracycline-resistant Escherichia coli on a dairy farm.

We examined how a dietary supplement affects the prevalence of antibiotic-resistant Escherichia coli on a dairy farm in Washington State. Between 2001 and 2004 the prevalence of fecal E. coli strains resistant to streptomycin, sulfadiazine, and tetracycline (SSuT strains) declined from 59.2% to 26.1% in the calf population. In 2003 the dairy discontinued use of a dietary supplement, and we hypothesized that the decline in prevalence of SSuT strains was related to this change in management. To test this we established three treatments in which calves received no supplement, the dietary supplement with oxytetracycline, or the dietary supplement without oxytetracycline. Calves receiving either dietary supplement had a significantly higher prevalence of SSuT E. coli than the no-supplement control group (approximately 37% versus 20%, respectively; P = 0.03). Importantly, there was no evidence that oxytetracycline contributed to an increased prevalence of fecal SSuT E. coli. We compared the growth characteristics of SSuT and non-SSuT E. coli in LB broth enriched with either the complete dietary supplement or its individual constituents. Both the complete dietary supplement and its vitamin D component supported a significantly higher cell density of SSuT strains (P = 0.003 and P = 0.001, respectively). The dry milk and vitamin A components of the dietary supplement did not support different cell densities. These results were consistent with selection and maintenance of SSuT E. coli due to environmental components independent of antibiotic selection.

Animals↗
PubMed2006

Antimicrobial drug resistance genes do not convey a secondary fitness advantage to calf-adapted Escherichia coli.

Maintenance of antimicrobial drug resistance in bacteria can be influenced by factors unrelated to direct selection pressure such as close linkage to other selectively advantageous genes and secondary advantage conveyed by antimicrobial resistance genes in the absence of drug selection. Our previous trials at a dairy showed that the maintenance of the antimicrobial resistance genes is not influenced by specific antimicrobial selection and that the most prevalent antimicrobial resistance phenotype of Escherichia coli is specifically selected for in young calves. In this paper we examine the role of secondary advantages conveyed by antimicrobial resistance genes. We tested antimicrobial-susceptible null mutant strains for their ability to compete with their progenitor strains in vitro and in vivo. The null mutant strains were generated by selection for spontaneous loss of resistance genes in broth supplemented with fusaric acid or nickel chloride. On average, the null mutant strains were as competitive as the progenitor strains in vitro and in newborn calves (in vivo). Inoculation of newborn calves at the dairy with antimicrobial-susceptible strains of E. coli did not impact the prevalence of antimicrobial-resistant E. coli. Our results demonstrate that the antimicrobial resistance genes are not responsible for the greater fitness advantage of antimicrobial-resistant E. coli in calves, but the farm environment and the diet clearly exert critical selective pressures responsible for the maintenance of antimicrobial resistance genes. Our current hypothesis is that the antimicrobial resistance genes are linked to other genes responsible for differential fitness in dairy calves.

Aging↗
PubMed2006

Using DNA microarrays to identify library-independent markers for bacterial source tracking.

Bacterial source tracking is used to apportion fecal pollution among putative sources. Within this context, library-independent markers are genetic or phenotypic traits that can be used to identify the host origin without a need for library-dependent classification functions. The objective of this project was to use mixed-genome Enterococcus microarrays to identify library-independent markers. Separate shotgun libraries were prepared for five host groups (cow, dog, elk/deer, human, and waterfowl), using genomic DNAs (gDNAs) from ca. 50 Enterococcus isolates for each library. Microarrays were constructed (864 probes per library), and 385 comparative genomic hybridizations were used to identify putative markers. PCR assays were used to screen 95 markers against gDNAs from isolates from known sources collected throughout the United States. This validation process narrowed the selection to 15 markers, with 7 having no recognized homologues and the remaining markers being related to genes involved in metabolic pathways and DNA replication. In most cases, each marker was exclusive to one of four Enterococcus species (Enterococcus casseliflavus, E. faecalis, E. hirae, or E. mundtii). Eight markers were highly specific to either cattle, humans, or elk/deer, while the remaining seven markers were positive for various combinations of hosts other than humans. Based on microarray hybridization data, the prevalence of host-specific markers ranged from 2% to 45% of isolates collected from their respective hosts. A 20-fold difference in prevalence could present challenges for the interpretation of library-independent markers.

Animals↗
PubMed2005

Polymorphisms in 16S rRNA genes of Flavobacterium psychrophilum correlate with elastin hydrolysis and tetracycline resistance.

Flavobacterium psychrophilum is the etiological agent of bacterial coldwater disease, which causes significant problems to aquaculture worldwide. A recent study (Soule M, Cain K, LaFrentz S, Call DR [2005] Infect Immun 73:3799-3802) identified two 16S rRNA gene sequence variants (6 base differences) within the variable stem-loop region 3 for F. psychrophilum strains ATCC 49418 and CSF 259-93. That study also hypothesized that F. psychrophilum is composed of at least 2 distinct genetic lineages (I and II) described by a microarray-based comparative genomics study. In the present study, we augmented an existing 16S rDNA microarray to detect both 16S rRNA sequence variants from F. psychrophilum. Subsequent microarray experiments showed that CSF 259-93 hybridized as expected, but ATCC 49418 was positive for both sequence variants. We then developed a PCR-restriction fragment length polymorphism (RFLP) assay (MnlI and MaeIII) to distinguish between the 2 sequences. Gel isolation of PCR-RFLP products, cloning, and sequencing confirmed that ATCC 49418 harbors both 16S rRNA sequences. Microarray experiments showed that 11 of 14 strains from genetic Lineage I harbor both the CSF 259-93 and ATCC 49418 16S rRNA sequence variants, whereas all 15 Lineage II strains were only positive for the CSF 259-93 sequence (p < 0.0001). Elastin hydrolysis and tetracycline resistance were most closely associated with the latter strains (p < 0.0001 and p = 0.024, respectively). These data support the hypothesis that F. psychrophilum is composed of at least 2 distinct genetic lineages that are closely associated with host origin.

Base Sequence↗
PubMed2005

Long oligonucleotide microarrays in wheat: evaluation of hybridization signal amplification and an oligonucleotide-design computer script.

A computer script was written in the Perl language to design equal-length long oligonucleotides from DNA sequences. The script allows the user to specify G + C content, melting temperature, self-complementarity, the maximum number of contiguous duplicate bases, whether to start with the first start codon and whether to report reverse complements. Microarrays were fabricated with 95 oligonucleotides (60 mers) representing 41 genes. The microarray was interrogated with cDNA from roots and shoots of two near-isogenic lines and a commercial cultivar of Triticum aestivum L. (hexaploid wheat) challenged with cold temperature, hot temperature, or the biological control bacterium Pseudomonas fluorescens. Self-complementarity of the oligonucleotides was negatively correlated with signal intensity in 23 of 54 arrays (39%; P <0.01). Tyramide signal amplification was essential for signal generation and detection. Genes involved in signal transduction pathways responded similarly following exposure to cold, heat and P. fluorescens, suggesting intersection of the pathways involved in response to these disparate stress factors. Microarray results were corroborated by quantitative real-time PCR in 75% of samples assayed. We conclude that long oligonucleotide microarrays for interrogation with cDNA from hexaploid wheat should be constructed from oligonucleotides having minimal self complementarity that also meet user-specified requirements of length, G + C content and melting temperature; multiple oligonucleotides should be used to represent each gene; and Tyramide signal amplification is useful in wheat oligonucleotide microarray studies.

Base Sequence↗
PubMed2005

Challenges and opportunities for pathogen detection using DNA microarrays.

DNA microarrays offer the potential for simultaneous detection of many pathogens that are of interest to homeland security, public health, medicine, and veterinary diagnostics. These tools are best suited for detecting the presence or absence of genetic sequences characteristic of specific pathogens, but microarrays are poorly suited for determining pathogen viability, and current methods provide only limited potential for pathogen enumeration. Two basic strategies have been described for pathogen detection: using enzymatic amplification to generate targets for interrogation with a microarray, or using direct interrogation of DNA or RNA without pre-amplification. Multiplex PCR has the advantage of a high degree of sensitivity and specificity, but associated microarrays are necessarily limited in scope. PCR-independent, whole-genome amplification eliminates biases inherent in PCR amplification and can accommodate more extensive microarrays, but assay sensitivity is compromised and these methods are probably of limited use when testing tissue samples. Direct hybridization of DNA or RNA provides the least bias in gene detection, but also the lowest level of analytic sensitivity. Ultimately, cost and limited sample throughput make it unlikely that planar microarrays will play a significant role in future pathogen detection schemes. Alternative microarray formats such as bead arrays, however, may circumvent the cost and throughput limitations and permit us to apply what we have learned from planar microarrays to develop robust pathogen detection systems. Assay validation and sample preparation will continue to be significant challenges for these detection systems.

Animals↗
PubMed2005

Prevalence and antimicrobial resistance of thermophilic Campylobacter spp. from cattle farms in Washington State.

The prevalence of thermophilic Campylobacter spp. was investigated in cattle on Washington State farms. A total of 350 thermophilic Campylobacter isolates were isolated from 686 cattle sampled on 15 farms (eight dairies, two calf rearer farms, two feedlots, and three beef cow-calf ranches). Isolate species were identified with a combination of phenotypic tests, hipO colony blot hybridization, and multiplex lpxA PCR. Breakpoint resistance to four antimicrobials (ciprofloxacin, nalidixic acid, erythromycin, and doxycycline) was determined by agar dilution. Campylobacter jejuni was the most frequent species isolated (34.1%), followed by Campylobacter coli (7.7%) and other thermophilic campylobacters (1.5%). The most frequently detected resistance was to doxycycline (42.3% of 350 isolates). Isolates from calf rearer facilities were more frequently doxycycline resistant than isolates from other farm types. C. jejuni was most frequently susceptible to all four of the antimicrobial drugs studied (58.8% of 272 isolates). C. coli isolates were more frequently resistant than C. jejuni, including resistance to quinolone antimicrobials (89.3% of isolates obtained from calves on calf rearer farms) and to erythromycin (72.2% of isolates obtained from feedlot cattle). Multiple drug resistance was more frequent in C. coli (51.5%) than in C. jejuni (5.1%). The results of this study demonstrate that C. jejuni is widely distributed among Washington cattle farms, while C. coli is more narrowly distributed but significantly more resistant.

Animal Husbandry↗
PubMed2005

Genetic diversity of Listeria monocytogenes strains from a high-prevalence dairy farm.

Listeria monocytogenes is a significant food-borne human and veterinary pathogen. Contaminated silage commonly leads to disease in livestock, but the pervasive nature of the bacterium can make it difficult to identify the source of infection. An investigation of bovine listeriosis that occurred on a Pacific Northwest dairy farm ("farm A") revealed that the clinical strain was closely related to fecal strains from asymptomatic cows, and that farm environment was heavily contaminated with a diversity of L. monocytogenes strains. In addition, the farm A clinical strain was closely related to clinical and environmental strains obtained 1 year prior from a second Northwest dairy farm ("farm B"). To investigate the source(s) of contamination on farm A, environmental samples were collected from farm A at two time points. Pulsed-field gel electrophoresis characterization of 538 isolates obtained from that farm identified 57 different AscI pulsovars. Fecal isolates obtained from individual cows were the most genetically diverse, with up to 94% of fecal samples containing more than one pulsovar. The maximum numbers of pulsovars and serotypes isolated from a fecal sample of one cow were 6 and 4, respectively. Serotype 1/2a was isolated most frequently at both time points. Microarray genotyping of bovine listeriosis, fecal, and silage strains from both farms identified four probes that differentiated listeriosis strains from environmental strains; however, no probe was common to both bovine listeriosis strains.

Animals↗
PubMed2005

Combining suppression subtractive hybridization and microarrays to map the intraspecies phylogeny of Flavobacterium psychrophilum.

Reciprocal subtractive libraries were prepared for two strains of Flavobacterium psychrophilum, one virulent and the other avirulent in a trout challenge model. Unique clones were sequenced and their distribution assessed among 34 strains. The analysis showed that F. psychrophilum is composed of two genetic lineages, possibly reflecting host specificity.

Flavobacterium↗
PubMed2005

Suspension microarray with dendrimer signal amplification allows direct and high-throughput subtyping of Listeria monocytogenes from genomic DNA.

Listeria monocytogenes is a significant cause of food-borne disease and mortality; therefore, epidemiological investigations of this pathogen require subtyping methods that are rapid, discriminatory, and reproducible. Although conventional microarray subtyping analysis has been shown to be both high resolution and genetically informative, it is still relatively low throughput and technically challenging. Suspension microarray technology eliminates the technical issues associated with planar microarrays and allows high-throughput subtyping of L. monocytogenes strains. In this study, a suspension array assay using dendrimer signal amplification allowed rapid and accurate serovar identification of L. monocytogenes strains using genomic DNA as a target. The ability to subtype genomic DNA without PCR amplification allows probes to be designed for many different regions within the bacterial genome and should allow high-resolution subtyping not possible with multiplex PCR.

Bacterial Typing Techniques↗
PubMed2004

Amplicon secondary structure prevents target hybridization to oligonucleotide microarrays.

DNA microarrays that are used as end-point detectors for PCR assays are typically composed of short (15-25 mer) oligonucleotide probes bound to glass. When designing these detectors, we have frequently encountered situations where a probe would not hybridize to its complementary, terminally labeled PCR amplicon. To determine if failures could be explained by general phenomenon such as secondary structure, we designed a microarray to detect eight regions of the Escherichia coli 16S rDNA gene. We then amplified eight amplicons of different lengths using a biotin conjugated, antisense primer. Amplicons were then hybridized to the microarray and detected using a combination of signal amplification and fluorescence. In most cases, probe sequences complementary to the 5' region of the amplified products (sense orientation) did not hybridize to their respective amplicon. We tested for positional bias and showed that a biotin conjugated sense primer mirrored the same probe failures. Nick translated products, however, hybridized to all probes. Because nick translation generates many labeled fragments of random length, we concluded that this method disrupted secondary structure that otherwise prevented the amplicons from hybridizing to their respective probes. We also show that nick translation does not compromise detector sensitivity even when used with long PCR amplicons (ca. 1.5 kbp). Despite the increased cost of the nick translation, we concluded that this labeling strategy will reduce the time needed to design new assays as well as avoid possible false negatives during field applications. Alternative labeling strategies are also discussed.

Artifacts↗
PubMed2004

Quantitative oligonucleotide microarray fingerprinting of Salmonella enterica isolates.

We report on a genome-independent microbial fingerprinting method using nucleic acid microarrays for microbial forensics and epidemiology applications and demonstrate that the microarray method provides high resolution differentiation between closely related microorganisms, using Salmonella enterica strains as the test case. In replicate trials we used a simple 192 probe nonamer array to construct a fingerprint library of 25 closely related Salmonella isolates. Controlling false discovery rate for multiple testing at alpha = 0.05, at least 295 of 300 pairs of S.enterica isolate fingerprints were found to be statistically distinct using a modified Hotelling T2 test. Although most pairs of Salmonella fingerprints are found to be distinct, forensic applications will also require a protocol for library construction and reliable microbial classification against a fingerprint library. We outline additional steps required to produce such a protocol.

Bayes Theorem↗
PubMed2004

Developmental control of titin isoform expression and passive stiffness in fetal and neonatal myocardium.

Developmental changes in contractile behavior are known to occur during fetal and postnatal heart development. In this study, we examined whether adaptations take place in titin. A range of species was used to evaluate titin isoform expression and altered function during cardiac muscle development. A novel titin exon microarray that allows all 363 titin exons to be monitored simultaneously was used for transcript studies. Results reveal expression of fetal titin isoforms, characterized by additional spring elements both in the tandem Ig and PEVK region of the molecule. At the protein level, the fetal cardiac isoform predominates in fetal and neonatal myocardium and gradually disappears during postnatal development with a time course that varies in different species. Passive myocardium, contrary to previous reports, was found to be less stiff in the neonate than in the adult. This can be explained by the unique spring composition of fetal cardiac titin expressed by the neonate. Changes in titin expression are likely to impact functional transitions and diastolic filling behavior during development of the heart.

Aging↗
PubMed2004

Detection of pathogenic Vibrio spp. in shellfish by using multiplex PCR and DNA microarrays.

This study describes the development of a gene-specific DNA microarray coupled with multiplex PCR for the comprehensive detection of pathogenic vibrios that are natural inhabitants of warm coastal waters and shellfish. Multiplex PCR with vvh and viuB for Vibrio vulnificus, with ompU, toxR, tcpI, and hlyA for V. cholerae, and with tlh, tdh, trh, and open reading frame 8 for V. parahaemolyticus helped to ensure that total and pathogenic strains, including subtypes of the three Vibrio spp., could be detected and discriminated. For DNA microarrays, oligonucleotide probes for these targeted genes were deposited onto epoxysilane-derivatized, 12-well, Teflon-masked slides by using a MicroGrid II arrayer. Amplified PCR products were hybridized to arrays at 50 degrees C and detected by using tyramide signal amplification with Alexa Fluor 546 fluorescent dye. Slides were imaged by using an arrayWoRx scanner. The detection sensitivity for pure cultures without enrichment was 10(2) to 10(3) CFU/ml, and the specificity was 100%. However, 5 h of sample enrichment followed by DNA extraction with Instagene matrix and multiplex PCR with microarray hybridization resulted in the detection of 1 CFU in 1 g of oyster tissue homogenate. Thus, enrichment of the bacterial pathogens permitted higher sensitivity in compliance with the Interstate Shellfish Sanitation Conference guideline. Application of the DNA microarray methodology to natural oysters revealed the presence of V. vulnificus (100%) and V. parahaemolyticus (83%). However, V. cholerae was not detected in natural oysters. An assay involving a combination of multiplex PCR and DNA microarray hybridization would help to ensure rapid and accurate detection of pathogenic vibrios in shellfish, thereby improving the microbiological safety of shellfish for consumers.

Animals↗