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Ryan M Huebinger,
Ming-Mei Liu,
Scot E Dowd,
Fernando A Rivera-Chavez,
John Boynton,
Curtis Carey,
Kenneth Hawkins,
Christian T Minshall,
Steven E Wolf,
Joseph P Minei,
Robert C Barber
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ABSTRACT: Abstract Background: We examined the microbiota of bronchoalveolar lavage (BAL) samples with next-generation sequencing (NGS) technology to determine whether its results correlate with those of standard culture methods or affect patient outcome or both. Methods: We collected BAL samples in the surgical intensive care unit (SICU) as part of the standard of care for intubated individuals who had a Clinical Pulmonary Infection Score (CPIS) ≥6 points. A portion of the BAL fluid was sequenced for the 16S region of ribosomal deoxyribonucleic acid (rDNA) with the Roche 454 FLX Titanium sequencer. Sequences were analyzed through a data-analysis pipeline to identify the appropriate taxonomic designation (∼species) of each 16s sequence. The bacterial microbiota of each BAL sample was compared with the bacteria identified in the sample through standard culture methods. Correlations between the taxonomic diversity of the microbiota and clinical outcome were examined through linear regression and Pearson correlation. Results: Bronchoalveolar lavage samples from 12 individuals in the SICU who had a CPIS ≥6 points were examined through 454 pyrosequencing. The number of phylotypes (∼species) in the samples ranged from 15 to 129. The number of phyla in the BAL samples ranged from 3 to 14. There was little correlation between the bacteria identified by NGS and those identified with standard culture methods. The same predominant bacterial strain was identified by both culture and sequencing in only a single sample. The correlation between patient days on a ventilator and the number of species in BAL samples was significant (r=0.7435, p=0.0056; r(2)=0.5528). Conclusions: Increasing diversity of the bacterial microbiota in BAL samples correlates with the duration of mechanical ventilation. Bacteria identified through standard culture methods were not well correlated with the findings of NGS.
Surgical Infections 05/2013; · 1.80 Impact Factor
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Fernando A Rivera-Chávez, Ryan M Huebinger,
Agnes Burris,
Ming-Mei Liu,
Joseph P Minei,
John L Hunt,
Brett D Arnoldo,
Robert C Barber,
E Alhan,
B Bigalke,
F M Kovar,
A Rouhiainen
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ABSTRACT: Background. The triggering receptor expressed in myeloid cells (TREM-1) is a key mediator in the activation of the local inflammatory response during lung infections. We aimed to evaluate the effect of a functionally relevant TREM-1 single nucleotide polymorphism within the exon 2 (A → T) on the development of pneumonia in burn patients. Objective. To determine whether a single nucleotide polymorphism (SNP) within the exon 2 (A → T) in the TREM-1 gene is associated with ventilator-associated pneumonia (VAP) in burn-injured patients. Methods. 540 patients with ≥10% total body surface area (TBSA) burn injuries or inhalation injury were prospectively enrolled. The influence of a polymorphism (A → T) in exon 2 of the TREM-1 gene was evaluated for association with increased risk of pneumonia by logistic regression analysis. Measurements and Main Results. 209 patients met criteria for VAP. Multivariate regression analysis showed that, after adjustment for potential confounders, we found that carriage of the TREM-1 T allele is associated with more than a 3-fold increased risk of VAP (OR 6.3, 95% CI 4–9). Conclusions. A TREM-1 single nucleotide polymorphism within the exon 2 (A → T) is associated with the development of pneumonia in burn patients.
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ABSTRACT: Evaluation of single nucleotide polymorphisms (SNPs) in the interleukin-10 promoter (-592 and -819) on risk for death after burn injury.
Association between the IL-10 SNPs and outcome after burn injury was evaluated in a cohort of 265 patients from Parkland Hospital, Dallas, TX with ≥ 15% TBSA burns without non-burn trauma (ISS ≤ 16), traumatic or anoxic brain injury or spinal cord injury, who survived >48 h under an IRB-approved protocol. Clinical data were collected prospectively and genotyping was conducted by TaqMan assay. Whole blood from 31 healthy volunteers was stimulated with LPS (100 ng/mL) to determine the level of IL-10 expression for each allele by enzyme-linked immunosorbent assay (ELISA).
After adjustment for percent total body surface area (TBSA) burned, inhalation injury, age, gender, and race/ethnicity, carriage of ‑592A and/or ‑819T was significantly associated (P = 0.014) with a decreased risk for death (adjusted odds ratio: 0.404; 95% CI: 0.197-0.829). As the candidate SNPs were in complete linkage disequilibrium, it was not possible to distinguish which allele was associated with decreased mortality risk. Age, inhalation injury, and full-thickness burn size were significantly associated with increased risk for death. In the LPS stimulated blood of healthy controls, carriage of the -592A and/or -819T allele demonstrated a trend for decreased levels of IL-10 (P = 0.079).
Carriage of the ‑592A and/or ‑819T allele in the IL-10 promoter appears to reduce the risk for death after burn injury.
Journal of Surgical Research 11/2010; 164(1):e141-5. · 2.25 Impact Factor
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ABSTRACT: Despite the general success of genome-wide association studies, much heritability remains unidentified in many disease states. Some of this 'missing' heritability may lie in epistatic interactions among multiple loci, which are typically ignored. We utilized a method for simultaneous evaluation of epistatic interactions between allelic variations within genes confined to a single pathway, which we have termed as pathway genetic load (PGL).
In separate analyses, we evaluated the risk for sepsis and for death associated with alleles at six loci in the TLR4 signaling and response pathway previously known or suspected to be linked to the development of sepsis after traumatic injury. We evaluated 155 patients with > or =15% TBSA burns and without significant non-burn trauma [ISS < or =16], traumatic or anoxic brain injury or spinal cord injury, who survived > 48 h post-admission. Clinical data were collected prospectively and candidate genotypes were determined by TaqMan assay.
After adjustment for burn size, inhalation injury, age, gender and race, PGL was associated with increased probability for complicated sepsis (aOR=1.59; 95%CI=1.11-2.29; p=0.011) and death (aOR=1.75; 95%CI=1.11-2.76; p=0.017).
Relative size and variability of aORs indicate greater power to detect genetic associations with PGL compared to the analysis of loci individually by multivariate logistic regression.
Burns: journal of the International Society for Burn Injuries 04/2010; 36(6):787-92. · 1.95 Impact Factor
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Ryan M. Huebinger,
Ruben Gomez,
Daphne McGee,
Ling-Yu Chang,
Jessica E. Bender,
Terence O'Keeffe,
Agnes M. Burris,
Susan M. Friese,
Gary F. Purdue,
John L. Hunt,
Brett D. Arnoldo,
Jureta W. Horton,
Robert C. Barber
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ABSTRACT: Impaired mitochondrial activity has been linked to increased risk for clinical complications after injury. Furthermore, variant mitochondrial alleles have been identified and are thought to result in decreased mitochondrial activity. These include a nonsynonymous mitochondrial polymorphism (T4216C) in the nicotinamide adenine dinucleotide dehydrogenase 1 gene (ND1), encoding a key member of complex I within the electron transport chain, which is found almost exclusively among Caucasians. We hypothesized that burn patients carrying ND1 4216C are less able to generate the cellular energy necessary for an effective immune response and are at increased risk for infectious complications. The association between 4216C and outcome after burn injury was evaluated in a cohort of 175 Caucasian patients admitted to the Parkland Hospital with burns covering greater than or equal to 15% of their total body surface area or greater than or equal to 5% full-thickness burns under an institutional review board-approved protocol. To remove confounding unrelated to burn injury, individuals were excluded if they presented with significant non-burn-related trauma (Injury Severity Score ≥16), traumatic or anoxic brain injury, spinal cord injury, were HIV/AIDS positive, had active malignancy, or survived less than 48 h postadmission. Within this cohort of patients, carriage of the 4216C allele was significantly associated by unadjusted analysis with increased risk for sepsis-related organ dysfunction or septic shock (P = 0.011). After adjustment for full-thickness burn size, inhalation injury, age, and sex, carriage of the 4216C allele was associated with complicated sepsis (adjusted odds ratio = 3.7; 95% confidence interval, 1.5-9.1; P = 0.005), relative to carriers of the T allele.
Shock 12/2009; 33(1):19-23. · 2.85 Impact Factor
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ABSTRACT: Numerous studies have linked impaired mitochondrial activity with increased risk for clinical complications after injury. Furthermore, a number of nonsynonymous polymorphisms have been identified within the mitochondrial genome that are believed to impair cellular respiration. These DNA variants include a nonsynonymous polymorphism (T4216C) in the NADH dehydrogenase 1 gene (ND1), which encodes a key member of Complex I of the electron transport chain. We hypothesized that trauma patients who carry the ND1 4216C allele may be less able to generate the cellular energy necessary to mount an effective immune response and are at increased risk for death as well as sepsis complicated by organ dysfunction or shock.
We enrolled a cohort of 136 patients admitted to the Parkland Hospital Surgical intensive care unit (ICU) with significant trauma (Injury Severity Score > or = 16), > or =16 years of age, and with a minimum intensive care unit stay of > or =24 hours under a protocol approved by the UTSW and Parkland IRBs. Patients with brain death, spinal cord injury, active malignancy, HIV/AIDS or who survived <48 hours after admission were excluded. Clinical data were collected prospectively and T4216C was genotyped by polymerase chain reaction-restriction fragment length polymorphism.
After multivariate adjustment for mechanism, severity of injury, units of packed red blood cells given in the first 24 hours, age, gender, and race/ethnicity, carriage of the 4216 C-allele was significantly associated with increased risk for sepsis complicated by organ dysfunction or septic shock (adjusted odds ratio [aOR] = 3.68; 95%CI: 1.17-11.52; p = 0.02) as well as death (aOR = 4.56; 95% CI: 1.05-19.79; p = 0.04), relative to carriers of the T-allele.
Carriage of the mitochondrial 4216C-allele increases the risk for infectious complications and death after severe trauma.
The Journal of trauma 03/2009; 66(3):850-7; discussion 857-8. · 2.48 Impact Factor
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ABSTRACT: Bowhead whales (Balaena mysticetus) experienced a severe demographic population bottleneck caused by commercial whaling that ceased in 1914. Aboriginal subsistence whale harvests have continued and are managed by the International Whaling Commission. In an effort to provide management advice for bowhead whales, 25 microsatellite loci were isolated from genomic DNA libraries. This panel of markers will be utilized to analyse stock structure hypotheses of current bowhead whale populations.
Molecular Ecology Resources 05/2008; 8(3):612-5. · 3.06 Impact Factor
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ABSTRACT: Steller sea lions (Eumetopias jubatus) are listed as an endangered species in western Alaska and have exhibited a significant population decline throughout their range. Eight microsatellite loci were isolated from genomic DNA libraries. In addition, all these markers were found to be variable in nine individuals of the California sea lion (Zalophus californicus). This panel of markers was developed to analyse population structure in Steller sea lions throughout their range.
Molecular Ecology Notes 10/2007; 7(6):1097 - 1099. · 2.38 Impact Factor
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ABSTRACT: The Komodo monitor (Varanus komodoensis) is a classic example of a species that has been fragmented into small isolated populations over a limited range. This species,
classified as endangered under Appendix I of the Convention on the International Trade of Endangered Species. We describe
10 novel species-specific microsatellite loci characterized in representatives from three of the endemic island populations.
One locus, 12HDZ820 appears to be fixed in one population at an allele size not found in the other two. This microsatellite
suite should be helpful in augmenting the marker selection currently available for Komodo Monitor population studies.
Conservation Genetics 07/2007; 8(4):1017-1020. · 1.61 Impact Factor
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ABSTRACT: Analysis of 33 microsatellite loci for bowhead whales, including 22 new highly reliable markers, suggests present or historical departures from panmixia in Bering-Chukchi-Beaufort Seas bowhead whales. Although these bowheads are clearly genetically distinct from bowheads in the Sea of Okhotsk, we find significant patterns of genetic inhomogeneity among the Bering-Chukchi-Beaufort Seas samples. These samples exhibit strong and widespread departure from Hardy-Weinberg equilibrium, including significant evidence of a birth year effect or a historical bottleneck consistent with gene drift after commercial exploitation or thousands of years earlier. There is also significant evidence that whales of detectably different ancestry intermingle during some spatio-temporal portions of the annual migration but partially segregate in other portions. The most notable such pattern is seen in migratory pulses passing Barrow in the fall. Estimates of F st associated with our findings of genetic structure in Bering-Chukchi-Beaufort Seas bowheads are extremely small compared to values for comparisons with the known separate stock in the Sea of Okhotsk, and are also smaller than values obtained by separating suspected familial lineages within the Bering-Chukchi-Beaufort Seas samples. Furthermore, potential model misspecification provokes skepticism about some detected patterns, notably including the temporal ones. When analysis is limited to the most trusted markers and samples, sensitivity analyses show that most of our findings vanish and that the main sources of genetic signal in these data are scoring errors, familial relations, and birth year. We conclude that Bering-Chukchi-Beaufort Seas bowheads may comprise a complex spatio-temporal aggregation of animals with mixed and variable ancestry with an unknown degree of nonrandom mating, whose degree of genetic inhomogeneity is significantly less than what is seen between spatially isolated stocks. Despite these intriguing and complex biological findings, we have found no convincing evidence that Bering-Chukchi-Beaufort Seas bowheads should be managed as more than one stock.
04/2007;
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ABSTRACT: Eight microsatellite loci were isolated from a genomic DNA library created from Grant's gazelle (Gazella granti). Observed and expected levels of heterozygosity were computed utilizing 20 individuals from a population in Central Kenya. Tests for Hardy–Weinberg equilibria were conducted and found that two of the eight loci deviated from equilibrium in this population. These markers were developed to analyse the genetic effects of culling and isolation on a game preserve in Kenya.
Molecular Ecology Notes 07/2006; 6(4):1150 - 1151. · 2.38 Impact Factor
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ABSTRACT: There are three Boid species endemic to Madagascar, which represent two genera (Acrantophis, Sanzinia). We have characterized 10 microsatellite loci that are polymorphic in all three species. These markers will provide a system to study population segregation, population diversity, gene flow, and many other parameters. This data will be useful in planning and implementing conservation plans for all three of these endangered Boid species.
Molecular Ecology Notes 04/2005; 1(1‐2):41 - 43. · 2.38 Impact Factor
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ABSTRACT: Sixteen polymorphic microsatellite markers with an average allele size of k = 4.3 are identified from a genomic plasmid library constructed for giraffe (Giraffa camelopardalis ssp.). Primer sequences and marker data are reported in tabular form. The markers were screened in a population of 25 Maasai giraffe (G. c. tippelskirchi) collected near the Athi River, Kenya. The average observed heterozygosity for each marker was 0.36 with an average expected heterozygosity of 0.535. Hardy–Weinberg deviations are reported from this population, which is suspected to be inbred. The markers will be used to screen the captive giraffe population for subspecific or hybrid classification.
Molecular Ecology Notes 10/2002; 2(4):531 - 533. · 2.38 Impact Factor
