Decay rates of zoonotic pathogens and viral surrogates in soils amended with biosolids and manures and comparison of qPCR and culture derived rates.

AIMS: The purpose of this study was to establish inactivation decay constants of foodborne pathogens and coliphage in clay and sandy soils for future "downstream" analyses such as quantitative microbial risk analysis and to compare cultivation-dependent and -independent (e.g. qPCR) methods. METHODS AND RESULTS: Salmonella enterica, Campylobacter jejuni, Listeria monocytogenes, Escherichia coli O157:H7, and Clostridium perfringens, were seeded together with MS2 and ØX174 phages, into three waste matrices (Class B biosolids, swine lagoon effluent, cattle manure), and phosphate buffered saline (PBS) as a control, and applied to two soil types (sandy loam, clay loam) using two management practices (incorporated, surface applied). S. enterica and L. monocytogenes inactivation rates were positively affected (e.g. slower rate) by solid wastes, while C. jejuni was quickly inactivated by day 7 regardless of waste type. The use of qPCR provided more conservative inactivation rates, with qPCR-based rates typically twice as slow as cultivation-based. The effect of soil type and management were less apparent as rates were variably affected. For instance, incorporation of waste negatively impacted (e.g. faster rate) inactivation of Salmonella when measured by qPCR, while the opposite was true when measured by cultivation. Inactivation rates were organism∗waste∗soil∗management dependent since the interactions of these main effects significantly affected most combinations. CONCLUSIONS: Class B biosolids and cattle manure most often slowed inactivation when measured by cultivation, but the complex interactions between variables and organism made sweeping conclusions difficult. On the contrary cultivation-independent inactivation rates were negatively affected by solid wastes. Inactivation rates developed by cultivation-dependent and -independent assays needs further scrutiny as interprerations can vary by orders of magnitude depending on the organism∗environment combination. SIGNIFICANCE AND IMPACT OF THE STUDY: This study compares decay rate data based on waste, soil, management and assay type which can be further used in risk assessments.

Cultivation and qPCR Detection of Pathogenic and Antibiotic-Resistant Bacterial Establishment in Naive Broiler Houses.

Conventional commercial broiler production involves the rearing of more than 20,000 broilers in a single confined space for approximately 6.5 wk. This environment is known for harboring pathogens and antibiotic-resistant bacteria, but studies have focused on previously established houses with mature litter microbial populations. In the current study, a set of three naive houses were followed from inception through 11 broiler flocks and monitored for ambient climatic conditions, bacterial pathogens, and antibiotic resistance. Within the first 3 wk of the first flock cycle, 100% of litter samples were positive for and , whereas was cultivation negative but PCR positive. Antibiotic resistance genes were ubiquitously distributed throughout the litter within the first flock, approaching 10 to 10 genomic units g. Preflock litter levels were approximately 10 CFU g for heterotrophic plate count bacteria, whereas midflock levels were >10 colony forming units (CFU) g; other indicators demonstrated similar increases. The influence of intrahouse sample location was minor. In all likelihood, given that preflock levels were negative for pathogens and antibiotic resistance genes and 4 to 5 Log lower than flock levels for indicators, incoming birds most likely provided the colonizing microbiome, although other sources were not ruled out. Most bacterial groups experienced a cyclical pattern of litter contamination seen in other studies, whereas microbial stabilization required approximately four flocks. This study represents a first-of-its-kind view into the time required for bacterial pathogens and antibiotic resistance to colonize and establish in naive broiler houses.

Using broiler litter and swine manure lagoon effluent in sawdust-based swine mortality composts: Effects on nutrients, bacteria, and gaseous emissions.

Disposition of mortalities challenges confined animal feeding operations (CAFOs), especially sow (farrowing) farms, which experience mortalities daily. Regulations and transportation costs may preclude incineration, landfill burial, and rendering; therefore, swine CAFOs in Mississippi in the Mid-South U.S. often compost mortalities. In this study, a farm-standard composting mix of sawdust (S) and water (W) was compared with mixes where N was supplied by broiler litter (L) and water was replaced with swine lagoon effluent (E). The objective was to assess the effects of these manure byproducts: 1) on nutrients and bacteria in composts destined for land application; and 2) on emissions of ammonia and greenhouse gases. Three replications of four mixes (SW, SLW, SE, SLE) were compared in microcosms comprising modified plastic recycling bins. The experiment was repeated three times in different seasons in one year. Mixes were compared for differences in temperature, water content, nutrients (C, N, P, K, Ca, Mg, Na, Mn, Fe, Cu, Zn), bacteria (Gram-, Gram+, Clostridium perfringens, Salmonella, Listeria, Escherichia coli), and emissions (NH3, CO2, CH4, N2O). Litter addition increased composting temperatures initially and after aerations; increased nutrient concentrations, except C, in start mixes and all except C and N, in finish mixes; increased Gram+ bacteria, Salmonella, and E. coli in start mixes, but only Gram+s in finish mixes; and increased emissions. Effluent addition increased early composting temperatures; had no effect on nutrients or bacteria, except increased C. perfringens in start, but not finish mixes; and had no effect on emissions. Nutrients in finish composts did not differ among mixes for N (average 3.3%), but litter composts had more P and K, and lower N:P than composts without litter. Improving mortality composting is of global importance as increasing livestock populations and intensive animal production systems require practical, safe, environmentally sound disposal of carcasses.

A new sampler for stratified lagoon chemical and microbiological assessments.

A sampler was needed for a spatial and temporal study of microbial and chemical stratification in a large swine manure lagoon that was known to contain zoonotic bacteria. Conventional samplers were limited to collections of surface water samples near the bank or required a manned boat. A new sampler was developed to allow simultaneous collection of multiple samples at different depths, up to 2.3 m, without a manned boat. The sampler was tethered for stability, used remote control (RC) for sample collection, and accommodated rapid replacement of sterile tubing modules and sample containers. The sampler comprised a PVC pontoon with acrylic deck and watertight enclosures, for a 12 VDC gearmotor, to operate the collection module, and vacuum system, to draw samples into reusable autoclavable tubing and 250-mL bottles. Although designed primarily for water samples, the sampler was easily modified to collect sludge. The sampler held a stable position during deployment, created minimal disturbance in the water column, and was readily cleaned and sanitized for transport. The sampler was field tested initially in a shallow fresh water lake and subsequently in a swine manure treatment lagoon. Analyses of water samples from the lagoon tests showed that chemical and bacterial levels, pH, and EC did not differ between 0.04, 0.47, and 1.0 m depths, but some chemical and bacterial levels differed between winter and spring collections. These results demonstrated the utility of the sampler and suggested that future manure lagoon studies employ fewer or different depths and more sampling dates.

Broiler litter ammonia emissions near sidewalls, feeders, and waterers.

Ammonia (NH3) volatilized from broiler litter diminishes indoor air quality, which can potentially decrease bird productivity. Emissions of NH3 exhausted from broiler houses pose environmental concerns for ecosystem biodiversity, aquatic nutrient enrichment, and particulate formation in the atmosphere. Research was conducted sampling litter (rice hull base) in 3 tunnel-ventilated commercial broiler houses during wk 3 (mid-growout) of 6 flocks. The purpose was to assess NH3 generated near the sidewalls, waterers, and feeders. Litter samples (100 g) were placed in chambers receiving constant air flow. Boric acid (H3BO3) titration each 24 h for 4 d was used to determine NH3 volatilized from the samples. Litter located near waterers emitted the most cumulative NH3 (approximately 12.3 mg of N•kg of litter(-1)•h(-1)) with less NH3 associated with feeders and sidewalls (2.9 to 7.6 mg of N•kg of litter(-1)•h(-1)). Moisture content of litter samples was greatest at waterers (45%) followed by sidewalls (26%) and feeders (20%). In addition, litter pH at the sidewalls and feeders could be predicted by linear equations associated with the number of flocks on the litter. At the waterers, litter pH was differentiated based on the half of house where higher litter pH existed in the nonbrood half (8.55 vs. 8.13). The results indicate that controlling NH3 near watering lines to a level consistent with feeding lines and near the house wall could reduce NH3 generated by 38 to 77%. These findings support efforts for NH3 control at mid-growout, especially considering zone litter treatments near waterers and appropriate attention to waterer management.

Runoff quality from no-till cotton fertilized with broiler litter in subsurface bands.

Surface broadcast of broiler litter to no-till row crops exposes the litter and its nutrients to risks of loss in runoff water and volatilization and may limit the potential benefit of litter to the crops. Subsurface banding of litter could alleviate these risks. A field study was conducted in 2008 and 2009 on an upland Falkner silt loam soil to determine the effect of broiler litter placement on runoff nutrient losses from no-till cotton ( L.). Treatments included surface broadcast broiler litter applied manually, subsurface-banded litter applied by tractor-drawn equipment, and no broiler litter, all in combination with or without winter wheat ( L.) cover crop residue. Broiler litter rate was 5.6 Mg ha. The experimental design was a randomized complete block with a split-plot arrangement of treatments replicated three times. In 2008, simulated rainfall was used to generate runoff 27 d after litter application. Subsurface-banded litter reduced runoff total C, N, P, NH, NO, Cu, Zn and water-soluble P (WP) concentrations by 72, 64, 51, 49, 70, 36, 65, and 77%, respectively, compared with surface broadcast. The reductions were greater in 2009 where runoff occurred 1 d after litter application. Bacterial runoff was decreased by one log with subsurface-banded litter compared to surface broadcast. Except for C, NH, N, and WP, the presence of winter cover crop residue did not affect the load or runoff nutrient concentrations in either year. The results indicate that subsurface banding litter to no-till cotton substantially reduces nutrient and bacterial losses in runoff compared with surface broadcasting.

The effect of poultry manure application rate and AlCl(3) treatment on bacterial fecal indicators in runoff.

Increasing costs associated with inorganic fertilizer have led to widespread use of broiler litter. Proper land application, typically limiting nutrient loss, is essential to protect surface water. This study was designed to evaluate litter-borne microbial runoff (heterotrophic plate count bacteria, staphylococci, Escherichia coli, enterococci, and Clostridium perfringens) while applying typical nutrient-control methods. Field studies were conducted in which plots with high and low litter rates, inorganic fertilizer, AlCl(3)-treated litter, and controls were rained on five times using a rain generator. Overall, microbial runoff from poultry litter applied plots was consistently greater (2-5 log(10) plot(-1)) than controls. No appreciable effect on microbial runoff was noted from variable litter application rate or AlCl(3) treatments, though rain event, not time, significantly affected runoff load. C. perfringens and staphylococci runoff were consistently associated with poultry litter application, during early rain events, while other indicators were unreliable. Large microbial runoff pulses were observed, ranging from 10(2) to 10(10) CFU plot(-1); however, only a small fraction of litter-borne microbes were recoverable in runoff. This study indicated that microbial runoff from litter-applied plots can be substantial, and that methods intended to reduce nutrient losses do not necessarily reduce microbial runoff.

Effect of surface incorporation of broiler litter applied to no-till cotton on runoff quality.

Surface application of broiler litter to no-till cotton could lead to degradation of water quality. Incorporation of broiler litter into the top surface soil (0.05 m) could alleviate this risk. A 2-yr field study was conducted on a silt loam upland soil to determine the effect of incorporation of broiler litter into the soil surface on nutrient and bacterial transport in runoff. The experimental design was a randomized complete block with four treatments and three replications. Treatments were (i) unfertilized control; (ii) surface-appliedbroiler litter at 7.8 Mg ha(-1) without incorporation; (iii) surface-applied broiler litter at 7.8 Mg ha(-1) with immediate incorporation; and (iv) inorganic fertilizer N (urea ammonium nitrate, 32% N) and inorganic fertilizer P (triple superphosphate) at the recommended rate. Phosphorus was surface appliedat 25 kg ha(-1) and N was injected at 101 kg ha(-1) into the soil using a commercial liquid fertilizer applicator. Runoff was collected from small runoff plots (2.4 m by 1.6 m) established at the bottom side of main plots (13.7 m by 6.0 m). Incorporation of broiler litter reduced total N (TN), NO3-N, water soluble P (WSP), and total P (TP) concentrations in runoffby 35, 25, 61, and 64%, respectively, and litter-associated bacteria by two to three orders of magnitude compared with unincorporated treatment. No significant difference in total suspended solids (TSS) in runoffwas obtained between incorporated and unincorporated treatments. Incorporation of broiler litter into the surface soil in the no-till system immediately after application minimized the potential risk for surface nutrient losses and bacteria transport in runoff.

Microbial and antibiotic resistant constituents associated with biological aerosols and poultry litter within a commercial poultry house.

Poultry are known to harbor antibiotic resistant and pathogenic bacteria, and as such poultry litter and poultry house air can be contaminated with these bacteria. However, the presence of antibiotic resistant bacteria in biological aerosols and litter is largely not understood. The purpose of this study was to determine the amount of aerosolized bacteria and endotoxin, particularly fecal indicators, staphylococci, and enterococci, associated with poultry house and outdoor air. Aerosol samples were collected at multiple locations on the farm and in the house. Antibiotic resistance was investigated using the Kirby Bauer method on selected isolates using twelve different antibiotics spanning both narrow to broad spectrums of effectiveness. Overall there was a cyclical increase in bacterial concentrations as flocks progressed from pre-flock to late-flock, with >2 orders magnitude lower concentration during pre-flock periods (no chickens), in both the litter and aerosol samples. The house environment provided for significantly concentrated bacterial and endotoxin levels. It was estimated that Staphylococcus bacteria accounted for at least 90% of cultured aerobic bacteria and culture-independent 16S rRNA analyses demonstrated that significant population changes occurred from pre- to late-flock. Rarely was an isolate resistant to more than 4 antibiotic classes; however there was a trend upwards in overall resistance of enterococci as the flock cycle progressed. It appears that although levels of antibiotic resistant bacteria were highly concentrated within the house, levels were much lower outside of the house, and very little house escape occurred.

Antibiotic resistant bacterial profiles of anaerobic swine lagoon effluent.

Although land application of swine (Sus scrofa) manure lagoon effluent is a common and effective method of disposal, the presence of antibiotic-resistant bacteria, both pathogenic and commensal can complicate already understood issues associated with its safe disposal. The aim of this study was to assess antibiotic resistance in swine lagoon bacteria from sow, nursery, and finisher farms in the southeastern United States. Effluents from 37 lagoons were assayed for the presence of Escherichia coli, Campylobacter, Listeria, and Salmonella. Antibiotic resistance profiles were determined by the Kirby-Bauer swab method for 12 antibiotics comprising eight classes. Statistical analyses indicated that farm type influenced the amount and type of resistance, with nurseries and sow farms ranking as most influential, perhaps due to use of more antibiotic treatments. Finisher farms tended to have the least amount of antibiotic class resistance, signaling an overall healthier market pig, and less therapeutic or prophylactic antibiotic use. Many bacterial isolates were resistant to penicillin, cephalosporin, and tetracycline class antibiotics, while nearly all were susceptible to quinolone antibiotics. It appeared that swine farm type had a significant association with the amount of resistance associated with bacterial genera sampled from the lagoons; nurseries contributed the largest amount of bacterial resistance.

Simple colorimetric microplate test of phage lysis in Salmonella enterica.

A simple microplate method, based on conversion of tetrazolium to formazan, was devised for rapidly assessing Salmonella survival after phage treatment. Results were easily interpretable. Monitoring with a microplate reader was useful, but not required. The method was used in defining phage-Salmonella interactions for selection of phage biocontrol cocktails.

Factors affecting iron sulfide-enhanced bacteriophage plaque assays in Salmonella.

Reaction of ferric ions with hydrogen sulfide (H(2)S) enhances contrast of phage plaques in H(2)S+ Salmonella, but contrast diminishes in weak H(2)S+ strains. H(2)S was affected by concentrations of peptones, glucose, ferric ammonium citrate (FAC) and sodium thiosulfate (ST), and by FAC:ST ratio, temperature, pH, air, and host strain. Increasing peptone levels was most important for improving contrast in weak H(2)S+ strains.

Isolation of salmonella bacteriophages from swine effluent lagoons.

Bacteriophages (phages) associated with Salmonella were collected from nine swine manure lagoons in Mississippi. Phages were isolated by an enrichment protocol or directly from effluent. For enrichment, chloroform-treated samples were filtered (0.22 mum) and selectively enriched by adding a cocktail of Salmonella strains in trypticase soy broth. After overnight incubation at 35 degrees C, chloroform was added and samples stored at 5 degrees C. Enriched samples were tested by double agar layer (DAL) plaque assay against individual Salmonella isolates. Phage titers of 2.9 x 10(8) to 2.1 x 10(9) plaque forming units (pfu) per mL were produced, but estimation of phage titers in lagoons was not possible. For direct isolation, effluent was clarified by centrifugation, filtered (0.22 microm), and used in DAL plaque assays to select single-plaque isolates for 15 Salmonella strains. Plaque counts varied among Salmonella strains and lagoons. The most sensitive strain for direct phage recovery was ATCC 13311. Phage titers estimated by direct isolation with ATCC 13311 ranged among lagoons from 12 to 148 pfu per mL. In limited host range tests, 66 isolates recovered by the enrichment protocol produced plaques only on Enteritidis and Typhimurium strains of Salmonella and none produced plaques on lagoon isolates of Citrobacter, Escherichia, Proteus, Providencia, or Serratia. Electron microscopy (EM) showed purified enrichment isolates had Podoviridae morphology (tailless 50-nm icosahedral heads with tail spikes). Electron microscopy of clarified concentrated effluent showed 5.5:1 tailless to tailed phages. The isolated phages have potential as typing reagents, specific indicators, and biocontrol agents of Salmonella.

Enhanced contrast of bacteriophage plaques in Salmonella with ferric ammonium citrate and sodium thiosulfate (FACST) and tetrazolium red (TZR).

Visualization of bacteriophage plaques may be enhanced by addition of ferric ammonium citrate and sodium thiosulfate (FACST) or 2,3,5-triphenyltetrazolium chloride (tetrazolium red, TZR) to the soft agar layer of a traditional bacteriophage plaque assay. Background color from these reagents improved contrast between clear plaques and turbid host lawns in trypticase soy agar (TSA) plates. Enhancement by FACST is based on reaction with hydrogen sulfide gas (H2S) produced by some strains of bacteria and was tested here using H2S+ and H2S- strains of Salmonella enterica subsp. enterica with a bacteriophage (Podoviridae) isolated from swine lagoon effluent. Only the H2S+ strain produced dark brown-black color in FACST-amended agar. Both strains showed bright pinkish-red color in TZR-amended agar. Color intensity for both reagents decreased with decreasing concentrations of the reagents. Contrast in FACST-amended plates appeared greater than that with TZR, but diminished after 12 h, while contrast in TZR-amended plates remained constant. At the concentrations tested, neither reagent affected plaque counts in the H2S+ strain. The FACST should be useful in bacteriophage plaque assays with H2S+ strains of Salmonella and other H2S+ bacteria.

Do current outcomes data support the technique of lumbar interbody fusion?

Over the past 20 years, significant technical advances in spinal surgery have made the concept of interbody fusion a reality. The procedure, whether approached anteriorly or posteriorly, appears to improve patient satisfaction and radiographic fusion rates in selected cases. The lack of a randomized, prospective study and difficulty in comparing outcomes between studies has made it difficult to draw such a conclusion from the literature.

C1-C2 transarticular screw fixation for atlantoaxial instability: a 6-year experience.

OBJECTIVE: We review a 6-year, single-center experience using the technique of C1-C2 transarticular screw fixation for atlantoaxial instability in 75 consecutive operations. METHODS: The study group was composed of 43 men and 32 women, with a mean age of 44 years (range, 8-76 yr). Each patient had documented atlantoaxial instability. In 28 patients (37%), atlantoaxial instability was a result of trauma; in 22 patients, (29%), it was a result of rheumatoid arthritis; in 16 patients (21%), it was a result of prior surgery; and in 9 patients (12%), it was a result of congenital abnormalities. All patients underwent stabilization with C1-C2 transfacetal screws and a posterior interspinous construct. Nine patients had unilateral screws placed. Postoperatively, the patients were maintained in a rigid cervical orthosis for a mean of 11 weeks (range, 8-15 wk); five patients were immobilized with halo fixation for a mean of 13 weeks (range, 10-16 wk). The mean follow-up period was 2.4 years (range, 1-5.5 yr). RESULTS: Osseous fusion was documented in 72 patients (96%). There were no hardware failures; however, three patients developed pseudarthrosis. Two superficial wound infections (one at the graft site and one at the cervical incision site) required antibiotic therapy. Four patients had transient suboccipital hypesthesia. No instances of an errant screw, dural laceration, or injury to the vertebral artery, spinal cord, or hypoglossal nerve were noted. CONCLUSION: C1-C2 transarticular screw fixation supplemented with an interspinous construct yielded a 96% fusion rate, with a low incidence of complications. We attribute our successful outcomes to careful preoperative assessment and meticulous surgical technique.

Threaded cortical bone dowels for lumbar interbody fusion: over 1-year mean follow up in 28 patients.

OBJECT: The authors retrospectively reviewed a series of 35 patients with mechanical low-back or one- to two-level discogenic pain; the patients underwent lumbar interbody fusion in which threaded cortical bone dowels (TCBDs) were placed to treat degenerative disc disease. The purpose of the study was to delineate fusion rates and outcome data in this series of patients. METHODS: The series was composed of 18 women and 17 men whose mean age was 46 years (range 17-76 years). There were nine active cigarette smokers. All patients presented with symptoms consistent with mechanical low-back or discogenic pain, and magnetic resonance imaging revealed degenerative changes related to disc collapse at one or two vertebral levels. For placement of the TCBDs, 23 patients underwent posterior lumbar interbody fusion (PLIF), whereas 12 patients underwent anterior lumbar interbody fusion (ALIF). In all except one patient undergoing PLIF, pedicle screw and rod constructs were used without posterolateral fusion. In all patients undergoing ALIF except one, TCBDs were used as stand-alone devices without supplemental fixation. At follow up the success of fusion was determined by static lumbar radiography and/or computerized tomography scanning. The degree of lumbar lordosis at the diseased level was measured immediately postoperatively and compared with that documented on follow-up radiological studies. Outcomes were assessed using a modified Prolo Scale. Excellent and good outcomes were considered satisfactory, and fair or poor outcomes were considered unsatisfactory. In 28 patients (eight ALIF and 20 PLIF) radiological and clinical follow-up data were considered adequate. The mean follow-up duration was 12.3 months. Overall satisfactory outcome was 60%; 70% satisfactory outcome was noted in PLIF patients and 38% in ALIF patients. Osseous fusion was present in 95% of the patients in the PLIF group and in 13% of those in the ALIF group. Complications included one L-5 nerve root injury and two postoperative wound infections, all in patients who underwent PLIF; in an ALIF patient lateral breakout of one implant occurred at 8 months postoperatively. CONCLUSIONS: Analysis of the mean 12.3 month follow-up data indicates that there is a dramatically higher fusion rate in PLIF compared with ALIF procedures when TCBDs are used. The authors believe that it is important to note that in all the PLIF procedures except one, supplemental pedicle screw/rod constructs were used, whereas in ALIF procedures no supplemental fixation was performed. The results thus suggest that TCBDs are best used in PLIF in conjunction with pedicle screw and rod constructs.

Interaction of the PhiHSIC virus with its host: lysogeny or pseudolysogeny?

The marine phage PhiHSIC has been previously reported to enter into a lysogenic relationship with its host, HSIC, identified as Listonella pelagia. This phage produces a variety of plaques on its host, including turbid and haloed plaques, from which lysogens were previously isolated. These lysogens were unstable during long-term storage at -80( degrees ) C and were lost. When HSIC was reinfected with phage PhiHSIC, pseudolysogen-like interactions between the phage and its host were observed. The cells (termed HSIC-2 or HSIC-2e) produced high viral titers (10(11) ml(-1)) in the absence of inoculating phage and yet reached culture densities of nearly 10(9) ml(-1). Prophages were not induced by mitomycin C or the polyaromatic hydrocarbon naphthalene in cells harboring such infections. However, such cells were homoimmune to superinfection. Colonies hybridized strongly with a gene probe from a 100-bp fragment of the PhiHSIC genome, while the host did not. Analysis of chromosomal DNA preparations suggested the presence of a chromosomally integrated prophage. Phage adsorption experiments suggested that HSIC-2 was adsorption impaired. Because of the chromosomal prophage integration and homoimmunity, we interpret these results to indicate that PhiHSIC establishes a lysogenic relationship with its host that involves an extremely high level of spontaneous induction. This could be caused by a weak repressor of phage production. Additionally, poor phage adsorption of HSIC-2 compared to the wild type probably helped maintain this pseudolysogen-like relationship. In many ways, pseudolysogenic phage-host interactions may provide a paradigm for phage-host interactions in the marine environment.

Evaluation of marine bacterial lysogens for development of a marine prophage induction assay.

The demonstrated relationship between carcinogenicity of a chemical compound in mammals and its tendency to cause prophage induction in bacteria provides a method for biologically based carcinogen screening. Because of the need for this type of screening and the abundance of lysogens in the marine environment, 14 isolates were evaluated for the degree of prophage induction in exponentially growing cultures in the presence of a known mutagen (0.5 microg/ml mitomycin C). Assays were performed both in liquid culture and in microtiter plates. Virus-like particles were enumerated by epifluorescence microscopy after staining with SYBR-Gold. Two isolates designated P94-4B3 (identified as Halomonas aquamarina) and P94-4S3 (identified as Pseudomonas aeruginosa) were further evaluated for potential use. Because of the rapid growth, larger size of its virus-like particles, and linear response to increasing dose of mitomycin C, the P. aeruginosa st. P94-4S3 was determined a better candidate for the marine prophage induction assay (MPIA). The Pseudomonas isolate was then used in several experiments for the development and optimization of the MPIA procedure. Initial screenings of the MPIA were also performed with selected environmental pollutants.

Single-stage anterior-posterior decompression and stabilization for complex cervical spine disorders.

OBJECT: To evaluate the applicability and safety of single-stage combined anterior-posterior decompression and fusion for complex cervical spine disorders, the authors retrospectively reviewed 72 consecutive procedures of this type performed using a uniform technique at a single center. METHODS: The indications for decompression and stabilization included: postlaminectomy kyphosis (15 patients), trauma (19 patients), spondylosis and congenital stenosis (32 patients), and ossification of the posterior longitudinal ligament (six patients). All patients underwent anterior cervical corpectomies in which allograft fibula and plates were placed, with 89% of patients undergoing two- or three-level procedures (range one-four levels). Lateral mass plating with autograft (morselized iliac crest) fusion was performed in all patients while the same anesthetic agent was still in effect. A hard cervical collar was used postoperatively in all patients (mean 13 weeks). All patients were followed for a minimum of 2 years (mean 29 months). Fusion was determined to be successful in all 72 patients (100%). Although the short-term morbidity rate reached 32%, the significant long-term morbidity rate was only 5%. At the 2-year follow-up examination, anterior cervical plate dislodgment was seen in one patient, and 16 of the 516 lateral mass screws implanted were observed to have partially backed out. However, there were no cases of nerve root injury, strut graft extrusion, or anterior plate or screw fracture. There were no clinically significant hardware complications and no patient required repeated operation. CONCLUSIONS: The combined single-stage anterior-posterior decompression, reconstruction, and instrumentation procedure represents a viable option in the treatment of a select group of patients with complex cervical spinal disorders. The technique provides immediate rigid stabilization of the cervical spine, prevents anterior plate failure or strut graft extrusion, and eliminates the need for halo immobilization postoperatively. Furthermore, a higher rate of fusion is achieved with this combined approach than with the anterior approach alone.