RUMINANT NUTRITION SYMPOSIUM: Use of genomics and transcriptomics to identify strategies to lower ruminal methanogenesis.

Globally, methane (CH4) emissions account for 40% to 45% of greenhouse gas emissions from ruminant livestock, with over 90% of these emissions arising from enteric fermentation. Reduction of carbon dioxide to CH4 is critical for efficient ruminal fermentation because it prevents the accumulation of reducing equivalents in the rumen. Methanogens exist in a symbiotic relationship with rumen protozoa and fungi and within biofilms associated with feed and the rumen wall. Genomics and transcriptomics are playing an increasingly important role in defining the ecology of ruminal methanogenesis and identifying avenues for its mitigation. Metagenomic approaches have provided information on changes in abundances as well as the species composition of the methanogen community among ruminants that vary naturally in their CH4 emissions, their feed efficiency, and their response to CH4 mitigators. Sequencing the genomes of rumen methanogens has provided insight into surface proteins that may prove useful in the development of vaccines and has allowed assembly of biochemical pathways for use in chemogenomic approaches to lowering ruminal CH4 emissions. Metagenomics and metatranscriptomic analysis of entire rumen microbial communities are providing new perspectives on how methanogens interact with other members of this ecosystem and how these relationships may be altered to reduce methanogenesis. Identification of community members that produce antimethanogen agents that either inhibit or kill methanogens could lead to the identification of new mitigation approaches. Discovery of a lytic archaeophage that specifically lyses methanogens is 1 such example. Efforts in using genomic data to alter methanogenesis have been hampered by a lack of sequence information that is specific to the microbial community of the rumen. Programs such as Hungate1000 and the Global Rumen Census are increasing the breadth and depth of our understanding of global ruminal microbial communities, steps that are key to using these tools to further define the science of ruminal methanogenesis.

The Complete Genome Sequence of Methanobrevibacter sp. AbM4.

Methanobrevibacter sp. AbM4 was originally isolated from the abomasal contents of a sheep and was chosen as a representative of the Methanobrevibacter wolinii clade for genome sequencing. The AbM4 genome is smaller than that of the rumen methanogen M. ruminantium M1 (2.0 Mb versus 2.93 Mb), encodes fewer open reading frames (ORFs) (1,671 versus 2,217) and has a lower G+C percentage (29% versus 33%). Overall, the composition of the AbM4 genome is very similar to that of M1 suggesting that the methanogenesis pathway and central metabolism of these strains are highly similar, and both organisms are likely to be amenable to inhibition by small molecule inhibitors and vaccine-based methane mitigation technologies targeting these conserved features. The main differences compared to M1 are that AbM4 has a complete coenzyme M biosynthesis pathway and does not contain a prophage or non-ribosomal peptide synthase genes. However, AbM4 has a large CRISPR region and several type I and type II restriction-modification system components. Unusually, DNA-directed RNA polymerase B' and B'' subunits of AbM4 are joined, a feature only previously observed in some thermophilic archaea. AbM4 has a much reduced complement of genes encoding adhesin-like proteins which suggests it occupies a ruminal niche different from that of M1.

Genome sequencing of rumen bacteria and archaea and its application to methane mitigation strategies.

Ruminant-derived methane (CH4), a potent greenhouse gas, is a consequence of microbial fermentation in the digestive tract of livestock. Development of mitigation strategies to reduce CH4 emissions from farmed animals is currently the subject of both scientific and environmental interest. Methanogens are the sole producers of ruminant CH4, and therefore CH4 abatement strategies can either target the methanogens themselves or target the other members of the rumen microbial community that produce substrates necessary for methanogenesis. Understanding the relationship that methanogens have with other rumen microbes is crucial when considering CH4 mitigation strategies for ruminant livestock. Genome sequencing of rumen microbes is an important tool to improve our knowledge of the processes that underpin those relationships. Currently, several rumen bacterial and archaeal genome projects are either complete or underway. Genome sequencing is providing information directly applicable to CH4 mitigation strategies based on vaccine and small molecule inhibitor approaches. In addition, genome sequencing is contributing information relevant to other CH4 mitigation strategies. These include the selection and breeding of low CH4-emitting animals through the interpretation of large-scale DNA and RNA sequencing studies and the modification of other microbial groups within the rumen, thereby changing the dynamics of microbial fermentation.

Rumen microbial (meta)genomics and its application to ruminant production.

Meat and milk produced by ruminants are important agricultural products and are major sources of protein for humans. Ruminant production is of considerable economic value and underpins food security in many regions of the world. However, the sector faces major challenges because of diminishing natural resources and ensuing increases in production costs, and also because of the increased awareness of the environmental impact of farming ruminants. The digestion of feed and the production of enteric methane are key functions that could be manipulated by having a thorough understanding of the rumen microbiome. Advances in DNA sequencing technologies and bioinformatics are transforming our understanding of complex microbial ecosystems, including the gastrointestinal tract of mammals. The application of these techniques to the rumen ecosystem has allowed the study of the microbial diversity under different dietary and production conditions. Furthermore, the sequencing of genomes from several cultured rumen bacterial and archaeal species is providing detailed information about their physiology. More recently, metagenomics, mainly aimed at understanding the enzymatic machinery involved in the degradation of plant structural polysaccharides, is starting to produce new insights by allowing access to the total community and sidestepping the limitations imposed by cultivation. These advances highlight the promise of these approaches for characterising the rumen microbial community structure and linking this with the functions of the rumen microbiota. Initial results using high-throughput culture-independent technologies have also shown that the rumen microbiome is far more complex and diverse than the human caecum. Therefore, cataloguing its genes will require a considerable sequencing and bioinformatic effort. Nevertheless, the construction of a rumen microbial gene catalogue through metagenomics and genomic sequencing of key populations is an attainable goal. A rumen microbial gene catalogue is necessary to understand the function of the microbiome and its interaction with the host animal and feeds, and it will provide a basis for integrative microbiome-host models and inform strategies promoting less-polluting, more robust and efficient ruminants.

Characterization of closely related lactococcal starter strains which show differing patterns of bacteriophage sensitivity.

AIMS: To characterize a group of closely related Lactococcus lactis subsp. lactis casein starter strains used commercially, which differ in their sensitivity to bacteriophages isolated from the same industrial environment. METHODS AND RESULTS: Nine strains of L. lactis, six of which had been used as starter cultures for lactic casein manufacture, were shown to be closely related by pulsed-field gel electrophoresis and total DNA profiles. Nineteen phages which propagated on one or more of these starter strains were isolated from industrial casein whey samples. The phages were all small isometric-headed and could be divided into five groups on the basis of host range on the nine strains. Most of the phages did not give a PCR product with primers designed to detect the two most common lactococcal small isometric phage species (936 and P335). The hosts could be divided into six groups depending on their phage sensitivity. Plasmids encoding genes for the cell envelope associated PI-type proteinase, lactose metabolism and specificity subunits of a type I restriction/modification system were identified. CONCLUSIONS: This work demonstrates how isolates of the same starter strain may come to be regarded as separate cultures because of their different origins, and how these closely related strains may differ in some of their industrially relevant characteristics. SIGNIFICANCE AND IMPACT OF THE STUDY: This situation may be very common among lactococci used as dairy starter cultures, and implies that the dairy industry worldwide depends on a small number of different strains.

Rapid analysis of a plasmid by hydrophobic-interaction chromatography with a non-porous resin.

A HPLC technique has been developed, based on hydrophobic-interaction Chromatography with a non-porous packing (TSKgel Butyl-NPR, Tosoh Biosep LLC), that allows separation of the open circular (nicked) and supercoiled forms of five DNA plasmids, ranging in size from 4 to 30 kilo base pairs (kb). The identity of the bands was determined through light scattering and gel electrophoresis. Several buffers, gradients, flow-rates and temperatures were evaluated in determining the optimum operating conditions for the separation. For all plasmids a reversed ammonium sulfate in phosphate buffer (pH 7.1) gradient was established. The chromatographic resolution between the supercoiled and nicked peaks was found to be a function of flow-rate and temperature. The resolution and the elution order did not vary with plasmid size, with the open-circular form always being eluted before the supercoiled form. Hydrophobic-interaction chromatography is a useful alternative to ion exchange or size exclusion for the chromatography of large plasmids, up to 30 kb.

Early extubation after cardiac surgery: emotional status in the early postoperative period.

OBJECTIVE: To compare the emotional state during the first 3 days after coronary artery surgery of patients who had undergone early versus conventional extubation. DESIGN: A prospective, randomized, controlled trial. SETTING: University hospital, single center. PARTICIPANTS: Eligible patients (n = 100) presenting for elective coronary artery surgery, randomized to an early extubation group or a conventional extubation group. INTERVENTIONS: Emotional status was measured by the Hospital Anxiety and Depression Scale (HAD), the Self Assessment Manikin (SAM), and the Multiple Affect Adjective Check List-Revised (MAACL-R). Tests were administered preoperatively and on the 1st and 3rd days postoperatively. MEASUREMENTS AND MAIN RESULTS: Of patients in the conventional extubation group, 30% showed moderate-to-severe depressive symptoms (HAD score >10) on day 3 postoperatively compared with 8% of patients in the early extubation group (p = 0.02). There was a clinically insignificant increase in MAACL-R depression score on the 1st postoperative day within both groups but no other differences within or between groups in SAM or MAACL-R scores. CONCLUSION: Early extubation results in fewer patients displaying depressive symptoms on the 3rd postoperative day but appears to have little effect on other measurements of emotional status. Anesthetic management during coronary artery bypass graft surgery may play an important role in the overall well-being of the patient by decreasing the incidence of postoperative depression.

Novel sucrose transposons from plant strains of Lactococcus lactis.

Lactococcus lactis strains isolated from vegetable products transferred the ability to ferment sucrose in conjugation experiments with the recipient strain L. lactis MG1614. Nisin production and sucrose fermentation were transferred together from two strains, but transfer also occurred from several other strains which did not produce nisin. Pulsed-field gel electrophoresis analysis showed that all transconjugants had acquired large chromosomal insertions at two main sites. Nisin sucrose transconjugants had gained inserts of 70 kb, while those that fermented sucrose without nisin production contained inserts of between 50 and 110 kb. Transconjugants from one donor had acquired a separate insertion of 55 kb which correlated with enhanced bacteriophage resistance, but contained neither nisin nor sucrose fermentation genes.

Early extubation following coronary artery bypass surgery: a prospective randomized controlled trial. The Fast Track Cardiac Care Team.

STUDY OBJECTIVES: To determine the safety of early extubation (EE) after coronary artery surgery. DESIGN: Prospective randomized controlled trial. SETTING: The cardiac surgery operating room and ICU of a university-affiliated teaching hospital. PATIENTS: One hundred eligible patients presenting for elective coronary artery surgery. INTERVENTIONS: Patients randomized to the EE group were administered a reduced dose of fentanyl (15 microg/kg) and an anesthetic compatible with EE, while patients randomized to the conventional extubation (CE) group were given fentanyl (50 microg/kg). MEASUREMENTS AND RESULTS: The time to extubation in the EE group (median, 240 min; range, 30 to 930 min) was significantly less than the CE group (median, 420 min; range, 125 to 1,140 min) (p<0.01). Twenty patients were withdrawn from the study according to protocol guidelines. There were no cases of reintubation or complications attributable to EE. CONCLUSIONS: By using an appropriate anesthetic technique and postoperative management, EE can be achieved following coronary artery bypass surgery without major complications.

Characterization of lactococci isolated from minimally processed fresh fruit and vegetables.

Lactic acid bacteria isolated from minimally processed fresh fruit and vegetable products were identified as Lactococcus lactis subsp. lactis on the basis of phenotypic tests, presence of lactococcal IS elements, and partial sequence analysis of the 16S rRNA gene. Isolated bacteria were differentiated using pulsed-field gel electrophoresis of SmaI digests of genomic DNA. Sprouted seeds were the best source of strains, and lactococci appear to be the dominant microflora on these products during the period they are intended to be eaten. Although these plant strains showed many similarities to strains of L. lactis used as dairy starter cultures, their carbohydrate fermentation patterns were unusual and probably reflect their environmental origin. Most strains fermented sucrose and xylose, and some also fermented raffinose and melibiose. Most of the bacteriocin-producing strains produced nisin, and nisin genes could also be detected in strains that showed no bacteriocin activity, or that produced a different bacteriocin with a narrow spectrum of activity. One strain produced nisin but was unable to ferment sucrose, properties that have been generally regarded as linked. These strains may have uses as biopreservatives for minimally processed plant products.

Early developmental destruction of terminals in the striatal target induces apoptosis in dopamine neurons of the substantia nigra.

Many developing neural systems with peripheral projections depend on their target for trophic support during a critical period of natural cell death. Much less is known about central systems. That dopaminergic neurons of the substantia nigra may depend on their target, the striatum, during development is suggested by the presence of a natural apoptotic cell death event in these neurons that can be augmented by an early developmental axon-sparing striatal injury. To further assess the target dependence of these neurons, we have used the selective neurotoxin 6-hydroxydopamine to lesion their terminals within the striatum during development, while sparing intrinsic striatal target neurons. This lesion results in an induction of apoptotic cell death in phenotypically defined dopaminergic neurons that appears on the third postlesion day and persists until the tenth. This inducibility of cell death is dependent on developmental age: it is most marked before postnatal day (PND) 14. As late as PND42, inducibility is still detectable but much less so. In addition, at day 42 the morphology of cell death changes and becomes nonapoptotic in some cells. We conclude that terminal injury during a critical period of postnatal development, like axon-sparing target injury, induces augmented apoptotic death in mesencephalic dopaminergic neurons. These results suggest that these neurons have a period of target dependence. Regulation of this dependence is likely to influence the mature adult number of dopaminergic neurons.

Apoptotic neuron death in rat substantia nigra induced by striatal excitotoxic injury is developmentally dependent.

We have previously observed that an axon-sparing lesion of the striatum during development is associated with an induction of apoptotic cell death in the substantia nigra (SN). We have postulated that the induced death is due to a loss of striatum-derived trophic support. In other paradigms of neural development, it is often found that a need for trophic support is primarily observed only during a critical development period. We have therefore examined the time course for early striatal lesion to induce cell death in substantia nigra. We find that induction of apoptotic cell death is largely restricted to the first 2 postnatal weeks. After that time, induction of death in SN pars compacta abates. In SN pars reticulata, apoptotic death also abates, but by postnatal day 28, a non-apoptotic morphology of death appears. Thus, induced apoptotic death in SN is restricted to a critical developmental period.

Isolation and characterization of bacteriocin-producing lactic acid bacteria from ready-to-eat food products.

Lactic acid bacteria isolated from a range of foods sold in ready-to-eat form were screened for bacteriocin production. Twenty-two bacteriocin-producing cultures were isolated from 14 of the 41 foods sampled. Bacteriocin-producing isolates from meat, fish and dairy products were Lactobacillus and Leuconostoc species typically found associated with these products. Most of these isolates gave only a narrow inhibitory spectrum although two showed activity against Listeria monocytogenes. Fruit and vegetable products gave a broader range of organisms but most of the bacteriocin-producing cultures were found to be strains of Lactococcus. Several lactococci produced a nisin-like activity, and showed a broad inhibitory spectrum against the indicator strains tested. The ease with which bacteriocin-producing strains could be isolated implies that they are already being safely consumed in food, and highlights the potential for using bacteriocin-producing cultures for biopreservation, especially in association with minimally processed products.

Initial qualitative evaluation of computed radiography in an intensive care unit.

The first computed radiography (CR) unit in Australia was installed at St Vincent's Hospital, Melbourne, in February 1994. An initial qualitative evaluation of the attitude of the intensive care unit (ICU) physicians to the CR unit was conducted in June 1994 by use of a survey. The results of the survey of ICU physicians indicated that images were available faster than under the previous system and that the use of the CR system was preferred to evaluate chest tubes and line placements. While it is recognized that a further detailed radiological evaluation of the CR system is required to establish the diagnostic performance of CR compared with conventional film, some comments on the implementation of the system and ICU physician attitudes to the CR system are put forward for consideration by other hospitals examining the possible use of CR systems.

Differentiation of dextran-producing Leuconostoc strains from fermented rice cake (puto) using pulsed-field gel electrophoresis.

Lactic acid bacteria were isolated from puto, a fermented rice cake consumed as a breakfast and snack food in the Philippines. The microflora was dominated by dextran-producing leuconostocs, and these were differentiated into four groups using pulsed-field gel electrophoresis of restriction enzyme digested chromosomal DNA, in conjunction with taxonomic tests. The four groups corresponded to the species Leuconostoc mesenteroides subsp. mesenteroides, Leuconostoc pseudomesenteroides, Leuconostoc citreum and Leuconostoc fallax. Several strains showed an unusual clumping phenotype, and two of these were capable of inhibiting other strains of lactic acid bacteria.

Clinical information systems and quality of care in the intensive care unit.

The introduction of new medical technology needs to be associated with the assessment of the value of this technology in patient care. Traditional outcome measures of quality in health care (such as survival, cost, and "quality of life") may be of little value in assessing quality of patient care at the bedside. A model of quality in bedside patient care is presented and used in the assessment of the impact of introducing a bedside information system on patient care quality.

Fluctuating and/or progressive sensorineural hearing loss in children.

Sensorineural hearing loss (SNHL) which is sudden in onset, fluctuating, and/or progressive complicates medical management, hearing aid selection, and individualized educational planning for a hearing-impaired child. In spite of multidisciplinary evaluation and intervention, a gradual decrease in auditory acuity may continue unabated in a significant number of cases. Intercurrent middle ear disease and inconsistent audiologic technique can account for threshold variation in some cases. The study population consisted of 229 children (132 boys; 97 girls) aged 1 to 19.9 years at first audiogram which revealed at least a mild degree of sensorineural hearing loss in one or both ears (35 unilateral), and who demonstrated threshold variation of 10 dB or more in at least one ear at one or more of the standard audiometric test frequencies (250, 500, 1000, 2000, 4000, and 8000 Hz) and were without concurrent middle ear disease (mean length of follow-up, 4.9 years; mean number of audiograms, 10.3). Of 365 ears demonstrating threshold variation of 10 dB or more, 22 (6%) had purely progressive losses without intercurrent upward fluctuation, 208 (57%) had fluctuating thresholds with gradually progressive losses, and 135 (37%) had intermittent threshold fluctuation without permanent deterioration. The probability of contralateral threshold fluctuation if one ear fluctuated was 0.91, while the probability of contralateral progressive SNHL if one ear progressed was 0.67. Demographic data, presumptive etiology, degree of initial SNHL, audiometric configuration, and symmetry of threshold variation were considered as potential predictors of the likelihood of threshold fluctuation and/or progression.

Comparison of Leuconostoc oenos Strains by Pulsed-Field Gel Electrophoresis.

Pulsed-field gel electrophoresis of chromosomal DNA digested with NotI or SfiI was used to differentiate individual strains of Leuconostoc oenos. L. oenos isolates with 13 different restriction digest patterns were detected in New Zealand wines undergoing malolactic fermentation. The average genome size was estimated to be 1,800 kb.

Middle ear disease in young children with sensorineural hearing loss.

Early identification of children with sensorineural hearing loss (SNHL), coupled with the provision of appropriate conventional amplification constitute important elements of a (re)habilitative strategy. Often overlooked, however, is the problem posed by recurrent and chronic middle ear disease in the child with coexisting SNHL. This study focuses on 437 (237 boys and 200 girls) consecutive patients with bilateral SNHL, in the moderate range or poorer, who were diagnosed prior to age 5 years at Boys Town National Research Hospital. The mean threshold shift with effusion was 25.0 dB at 250 Hz, 28.5 dB at 500 Hz, 29.5 dB at 1000 Hz, 24.5 dB at 2000 Hz, and 27.5 dB at 4000 Hz. During a median follow-up period of 3.21 years, 154 (35.2%) of these children required surgical placement of tympanostomy tubes because of the severity of middle ear disease and its impact on auditory acuity. Accurate determination of the degree and audiologic configuration of a SNHL may be seriously hindered by a coexisting middle ear effusion. If initial findings indicate the presence of a middle ear effusion, measures of auditory function must be repeated after the effusion has been resolved by medical and/or surgical intervention.