Comparison of Acute Physiology and Chronic Health Evaluation (APACHE) II and American College of Surgeons National Surgical Quality Improvement Program (ACS-NSQIP) scoring system in predicting postoperative mortality in patients undergoing emergency laparotomy: A retrospective study.

Background and Aims: There is paucity of studies on preoperative risk assessment tools in patients undergoing emergency surgery. The present study evaluated the performance of the Acute Physiology and Chronic Health Evaluation (APACHE) II, American College of Surgeons National Surgical Quality Improvement Program (ACS-NSQIP) surgical risk calculator and American Society of Anesthesiologists (ASA) physical status (PS) classification system in patients undergoing emergency exploratory laparotomy. Methods: This retrospective study included 60 adult patients who underwent emergency exploratory laparotomy for perforation peritonitis. The clinical details, ASA PS classification, laboratory investigations and postoperative course of patients were retrieved from their medical records. Based on these details, APACHE II and ACS-NSQIP were calculated for the patients. The study's primary outcome was the accuracy of the preoperative APACHE II, ACS-NSQIP risk calculator and ASA PS class in predicting the postoperative 30-day mortality of patients. Results: The area under the curve (AUC) of APACHE II, ACS-NSQIP score, and ASA PS classification for mortality 30 days after surgery was 0.737, 0.694 and 0.601, respectively. The P value for the Hosmer-Lemeshow (H-L) test of scoring systems was 0.05, 0.25 and 0.05, respectively. AUC for postoperative complications was 0.799 for APACHE II, 0.683 for ACS-NSQIP and 0.601 for ASA PS classification. H-L test of these scoring systems for complications after surgery revealed P values of 0.62, 0.36 and 0.53, respectively. Conclusion: Compared to the ACS-NSQIP and ASA PS classification system, the APACHE II score has a better discriminative ability for postoperative complications and mortality in adult patients undergoing emergency exploratory laparotomy.

Development of a multiplex qPCR assay for the simultaneous detection of Mycoplasma bovis, Mycoplasma species, and Acholeplasma laidlawii in milk.

Contagious bovine mastitis caused by Mycoplasma bovis and other Mycoplasma species including Mycoplasma californicum, Mycoplasma bovigenitalium, Mycoplasma alkalescens, Mycoplasma arginini, and Mycoplasma canadense is an economical obstacle affecting many dairy herds throughout California and elsewhere. Routine bacteriological culture-based assays for the pathogens are slow and subject to false-positive results due to the presence of the related, non-pathogenic species Acholeplasma laidlawii. To address the need for rapid and accurate detection methods, a new TaqMan multiplex, quantitative real-time PCR (qPCR) assay was developed that targets the 16S rRNA gene of Mycoplasma, rpoB gene of M. bovis, and the 16S to 23S rRNA intergenic transcribed spacer (ITS) region of A. laidlawii. qPCR amplification efficiency and range of detection were similar for individual assays in multiplex as when performed separately. The multiplex assay was able to distinguish between M. bovis and A. laidlawii as well as detect Mycoplasma spp. collectively, including Mycoplasma californicum, Mycoplasma bovigenitalium, Mycoplasma canadense, Mycoplasma arginini and Mycoplasma alkalescens. In milk, the lower limit of detection of M. bovis, M. californicum, and A. laidlawii with the multiplex assay was between 120 to 250 colony forming units (CFU) per mL. The assay was also able to simultaneously detect both M. bovis and A. laidlawii in milk when present in moderate (103 to 104 CFU/mL) to high (106 to 107 CFU/mL) quantities. Compared to laboratory culture-based methods, the multiplex qPCR diagnostic specificity (Sp) was 100% (95% CI [86.8-100]; n = 26) and diagnostic sensitivity (Se) was 92.3% (95% CI [74.9-99.1]; n = 26) for Mycoplasma species in milk samples collected from California dairy farms. Similarly, the Sp was 100% (95% CI [90.5-100]; n = 37) and Se was 93.3% (95% CI [68.1-99.8]; n = 15) for M. bovis. Our assay can detect and distinguish among M. bovis, other prevalent Mycoplasma spp., and non-pathogenic Acholeplasma laidlawii for effective identification and control of mycoplasma mastitis, ultimately supporting dairy cattle health and high-quality dairy products in California.

Functionalization of multiwalled carbon nanotubes for enzyme immobilization.

A simple strategy for enzyme immobilization onto homo- and hetero-functionalized multiwalled carbon nanotubes (MWCNTs) is described. Homo-functionalization of MWCNTs can be carried out using an amino-silane compound, i.e., 3-aminopropyl-triethoxysilane (APTES). It is an important silane coupling agent, which promotes interfacial behavior of nanomaterials. Whereas, hetero-functionalization of MWCNTs can be performed using APTES and cross-linking agent glutaraldehyde (GA). Key parameters for the immobilization of an enzyme on MWCNTs are selection of an efficient functionalizing agent, sonication of MWCNTs, enzyme load and enzyme coupling time. The optimal level of these factors is very important to obtain high specific activity and immobilization efficiency of the developed immobilized biocatalyst. Both homo- and hetero-functionalized MWCNTs can be used to develop an efficient immobilized biocatalyst having good operational stability. However, hetero-functionalized MWCNTs are more potent candidate for enzyme immobilization. The present methodology provides very efficient approach for enzyme immobilization on a versatile support to achieve high enzyme selectivity and operational stability for various industrial applications.

Fructose production from inulin using fungal inulinase immobilized on 3-aminopropyl-triethoxysilane functionalized multiwalled carbon nanotubes.

The main objective of the present work was to modify multiwalled carbon nanotubes (MWCNTs) using 3-aminopropyl-triethoxysilane (APTES) to generate amino-terminated surfaces for inulinase immobilization, which can be further used for fructose production. CCRD of response surface methodology was used for optimization of inulinase immobilization on MWCNTs. At optimized parameters (APTES concentration 4%; sonication time 4 h; enzyme coupling time 1.5 h and enzyme load 15 IU), maximal inulinase activity and immobilization yield was 60.7% and 74.4%, respectively. Immobilized inulinase showed same pH optima of free enzyme, while an elevation in temperature optima to 60 °C was observed after its immobilization. Immobilized inulinase also shown enhancement in pH stability and thermostability. Overall, 4.54-fold rise in half-life of inulinase was detected after immobilization at 60 °C. Km and Vmax of inulinase decreased after immobilization. Immobilized inulinase preserved 28% of its residual activity after 10 consecutive batch cycles of inulin hydrolysis for the production of fructose.

Purification and characterization of two isoforms of exoinulinase from Penicillium oxalicum BGPUP-4 for the preparation of high fructose syrup from inulin.

Two exoinulinases, Exo-I and Exo-II from the culture broth of Penicillium oxalicum BGPUP-4 was purified using three-step purification method i.e., isopropanol precipitation, Q-Sepharose and Sephadex G-100 column chromatography. The molecular weight of Exo-I and Exo-II was determined to be 64.85 kDa and 32.54 kDa, respectively using MALDI-TOF. Exo-I and Exo-II showed high specificity for inulin and their respective Vmax/Km ratio was 3.74 and 7.20. Besides, both the inulinases also displayed specificity for lactose, sucrose and raffinose. Exo-I and Exo-II were stable at a pH range of 4.0-8.0 with pH optima 5.0. Optimal temperature for both the inulinases was 55 °C, and both the isoforms retained approximately 50% of their activity up to 70 °C. Ag+, Hg2+, Ba2+, Cu2+ and Ca2+ ions shown stimulatory effect on inulinases activity, while Fe2+, Mn2+, Co2+and EDTA completely inhibited enzyme activity. Purified enzyme was successfully used for the preparation of high fructose syrup from inulin.

Response surface optimization of solid state fermentation for inulinase production from Penicillium oxalicum using corn bran.

Response surface methodology has been implemented for the utilization of corn bran for inulinase production by Penicillium oxalicum. CCRD of RSM with 15 runs was practiced to optimize three independent variables: moisture (70-90%), incubation time (4-8 days) and pH (5-8). However, other media constituents viz. inulin (1%), NaNO3 (0.2%), NH4H2PO4 (0.2%), KH2PO4 (0.2%), MgSO4·7H2O (0.05%) and FeSO4·7H2O (0.001%) were kept constant during solid state fermentations. Solid state fermentations were carried out at 30 °C at flask-level. A substantial inulinase production (77.95 IU/gds) was obtained under the optimized conditions i.e., moisture (80%), incubation time (6.0 days) and pH (6.5). Multiple correlation coefficient 'R2' for inulinase production was 1.00, which justifies good agreement between experimental and predicted values. Besides, 'R2' value close to one, also authenticates the validity of the model. The experimentation carried out at laboratory scale shown corn bran a good substrate for inulinase production by P. oxalicum.

Solid-State Fermentation of Carrot Pomace for the Production of Inulinase by Penicillium oxalicum BGPUP-4.

Inulinases are an important class of industrial enzymes which are used for the production of high-fructose syrup and fructooligosaccharides. Inulin, a polyfructan, is generally employed for the production of inulinase, which is a very expensive substrate. A number of agroindustrial residues have been used for cost-effective production of inulinases. In the present study, carrot pomace was selected as a substrate for the production of inulinase by Penicillium oxalicum BGPUP-4 in solid-state fermentation. Carrot pomace is one of the good substrates for bioprocesses, because it is rich in soluble and insoluble carbohydrates. A central composite rotatable design (CCRD) used in response surface methodology was employed for the optimal production of inulinase from carrot pomace. Using CCRD, 15 runs were practiced to optimize the range of three independent variables: moisture content (70-90%), incubation time (4-6 days) and pH (5.0-7.0) for inulinase production. Carrot pomace supplemented with 0.5% inulin as an inducer, 0.2% NH4H2PO4, 0.2% NaNO3, 0.2% KH2PO4, 0.05% MgSO4·7H2O and 0.001% FeSO4·7H2O was used for the production of inulinase in solid-state fermentation at 30 °C. Inulinase production (322.10 IU per g of dry substrate) was obtained under the optimized conditions, i.e. moisture content of 90%, incubation time 4 days and pH=7.0. The corresponding inulinase/invertase (I/S) ratio (3.38) was also high, which indicates the inulolytic nature of the enzyme. Multiple correlation coefficients R for inulinase production and I/S ratio were 0.9995 and 0.9947, respectively. The R value very close to one indicates an excellent correlation between experimental and predicted results.

Biocatalytic strategies for the production of high fructose syrup from inulin.

The consumption of natural and low calorie sugars has increased enormously from the past few decades. To fulfil the demands, the production of healthy sweeteners as an alternative to sucrose has recently received considerable interest. Fructose is the most health beneficial and safest sugar amongst them. It is generally recognised as safe (GRAS) and has become an important food ingredient due its sweetening and various health promising functional properties. Commercially, high fructose syrup is prepared from starch by multienzymatic process. Single-step enzymatic hydrolysis of inulin using inulinase has emerged as an alternate to the conventional approach to reduce complexity, time and cost. The present review, outlines the enzymatic strategies used for the preparation of high fructose syrup from inulin/inulin-rich plant materials in batch and continuous systems, and its conclusions.

Sequential statistical optimization of lactose-based medium and process variables for inulinase production from Penicillium oxalicum BGPUP-4.

A statistical tool of response surface methodology was used sequentially to optimise lactose-based medium and process variables for inulinase production from Penicillium oxalicum BGPUP-4. Two-level CCRD with four variables, for each design, was used for the optimization study. The independent variables: lactose 1-3%, NH4H2PO4 0.2-0.5%, NaNO3 0.2-0.5%, pH 5.0-7.0 (design-1); temperature 25-35 °C, incubation time 4.0-6.0 days, inoculum size 1.0-3.0 mycelial agar discs and agitation 100-200 rpm (design-2) were selected for the present investigation. The optimised medium variables (lactose 3.70%, NH4H2PO4 0.35%, NaNO3 0.35% and pH 6.0) produced 44.44 (IU/ml) and 0.38 (g dry wt./50 ml) of inulinase and biomass yield, respectively. Thereafter, the optimization of process conditions (temperature 25 °C, incubation time 5 days, inoculum size 2 mycelial agar discs and agitation 150 rpm), increased the inulinase production 50.45 (IU/ml) and biomass yield 0.26 (g dry wt./50 ml). The good agreement between experimental and predicted values in both the designs, the coefficient of determination (R2 ) greater than 0.90 and very close to 1.0 shows an appropriate fitness of the polynomial quadratic models.

A panorama of bacterial inulinases: Production, purification, characterization and industrial applications.

Inulinases are important hydrolysing enzymes which specifically act on ß-2, 1 linkages of inulin to produce fructose or fructooligosaccharides. Fungi, yeasts and bacteria are the potent microbial sources of inulinases. The data on bacterial inulinases is scarce as compared to other microbial sources. Inulinases yield from bacteria is very less as compared to fungal and yeast sources of inulinases. Submerged fermentation (SmF) is the method of choice for the production of inulinases from bacterial sources. Moreover, inulin is a potent substrate for the production of inulinases in SmF. Many bacterial inulinases have been reported to display magnificent environment abiding features and variability in their biophysical and biochemical properties. These properties have attracted intention of many researchers towards exploring adverse ecological niches for more distinctive inulinase producing bacterial strains. Inulinases are substantially important in current biotechnological era due to their numerous industrial applications. High fructose syrup and fructooligosaccharides are two major industrial applications of inulinases. Additionally, there are many reports on the production of various metabolites like citric acid, lactic acid, ethanol, biofuels, butanediol etc. using mixed cultures of inulinase producing organisms with other microorganisms. The present review mainly envisages inulinase producing bacterial sources, inulinase production, purification, characterization and their applications.

Hypervariable pili and flagella genes provide suitable new targets for DNA high-resolution melt-based genotyping of dairy Geobacillus spp.

Although nonpathogenic in nature, spores of Geobacillus are able to attach to surfaces, germinate, and form biofilms, allowing rapid multiplication and persistence within milk powder processing plants, causing final product contamination, and eventually leading to a loss of revenue in terms of downgraded product quality. As a result, Geobacillus spp. have been found to be common contaminants of milk powder worldwide. Genotyping methods can help in gaining insight into the ecology and transmission of these thermophilic bacteria within and between dairy processing plants. The objective of this study was to use the assembled draft genomes of two Geobacillus spp. to identify and test new hypervariable genotyping targets for differentiating closely related dairy Geobacillus isolates. The two Geobacillus spp. strains obtained from high spore count powders were obtained in 2010 (isolate 7E) and in 1995 (isolate 126) and were previously shown to be of same genotype based on a variable number tandem repeat genotyping method. Significant nucleotide sequence variation was found in genes encoding pili and flagella, which were further investigated as suitable loci for a new high-resolution melt analysis (HRMA)-based genotyping method. Three genes encoding pulG (containing prepilin-type N-terminal cleavage domain), pilT (pili retraction protein), and fliW (flagellar assembly protein) were selected as targets for the new pili/flagella gene (PilFla) HRMA genotyping method. The three-gene-based PilFla-HRMA genotyping method differentiated 35 milk powder Geobacillus spp. isolates into 19 different genotype groups (D = 0.93), which compared favorably to the previous method (which used four variable number tandem repeat loci) that generated 16 different genotype groups (D = 0.90). In conclusion, through comparative genomics of two closely related dairy Geobacillus strains, we have identified new hypervariable regions that prove to be useful targets for highly discriminatory genotyping.

Rapid identification of dairy mesophilic and thermophilic sporeforming bacteria using DNA high resolution melt analysis of variable 16S rDNA regions.

Due to their ubiquity in the environment and ability to survive heating processes, sporeforming bacteria are commonly found in foods. This can lead to product spoilage if spores are present in sufficient numbers and where storage conditions favour spore germination and growth. A rapid method to identify the major aerobic sporeforming groups in dairy products, including Bacillus licheniformis group, Bacillus subtilis group, Bacillus pumilus group, Bacillus megaterium, Bacillus cereus group, Geobacillus species and Anoxybacillus flavithermus was devised. This method involves real-time PCR and high resolution melt analysis (HRMA) of V3 (~70 bp) and V6 (~100 bp) variable regions in the 16S rDNA. Comparisons of HRMA curves from 194 isolates of the above listed sporeforming bacteria obtained from dairy products which were identified using partial 16S rDNA sequencing, allowed the establishment of criteria for differentiating them from each other and several non-sporeforming bacteria found in samples. A blinded validation trial on 28 bacterial isolates demonstrated complete accuracy in unambiguous identification of the 7 different aerobic sporeformers. The reliability of HRMA method was also verified using boiled extractions of crude DNA, thereby shortening the time needed for identification. The HRMA method described in this study provides a new and rapid approach to identify the dominant mesophilic and thermophilic aerobic sporeforming bacteria found in a wide variety of dairy products.

Genotyping of dairy Bacillus licheniformis isolates by high resolution melt analysis of multiple variable number tandem repeat loci.

In dairy foods, the sporeformer Bacillus licheniformis can be the cause of spoilage or specification compliance issues. Currently used methods for genotyping B. licheniformis have limited discrimination with only 2 or 3 different subgroups being identified. Here, we have developed a multi-locus variable number tandem repeat analysis (MLVA) method and combined it with high resolution melt analysis (MLV-HRMA) for genotyping B. licheniformis. Five repetitive loci were identified and used as markers for genotyping 52 isolates from two milk powder processing plants and retail samples. Nineteen genotypes could be identified using both MLVA and MLV-HRMA leading to Hunter-Gaston discrimination indices (D-value) of 0.93 each. It was found that all 5 MLVA loci were stable following 10 days of sub-culturing of 8 representative isolates. All isolates were also genotyped using previously used methods including randomly amplified polymorphic DNA-PCR (RAPD) and partial rpoB sequencing. Five different RAPD profiles and 5 different partial rpoB sequence types were identified resulting in corresponding D-values of 0.6 and 0.46, respectively. Analysis of the genotypes from dairy samples revealed that dairy B. licheniformis isolates are more heterogeneous than previously thought and that this new method can potentially allow for more discriminatory tracking and monitoring of specific genotypes.

Genotyping of present-day and historical Geobacillus species isolates from milk powders by high-resolution melt analysis of multiple variable-number tandem-repeat loci.

Spores of thermophilic Geobacillus species are a common contaminant of milk powder worldwide due to their ability to form biofilms within processing plants. Genotyping methods can provide information regarding the source and monitoring of contamination. A new genotyping method was developed based on multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) in conjunction with high-resolution melt analysis (MLV-HRMA) and compared to the currently used method, randomized amplified polymorphic DNA PCR (RAPD-PCR). Four VNTR loci were identified and used to genotype 46 Geobacillus isolates obtained from retailed powder and samples from 2 different milk powder processing plants. These 46 isolates were differentiated into 16 different groups using MLV-HRMA (D = 0.89). In contrast, only 13 RAPD-PCR genotypes were identified among the 46 isolates (D = 0.79). This new method was then used to analyze 35 isolates obtained from powders with high spore counts (>10(4) spores · g(-1)) from a single processing plant together with 27 historical isolates obtained from powder samples processed in the same region of Australia 17 years ago. Results showed that three genotypes can coexist in a single processing run, while the same genotypes observed 17 years ago are present today. While certain genotypes could be responsible for powders with high spore counts, there was no correlation to specific genotypes being present in powder plants and retailed samples. In conclusion, the MLV-HRMA method is useful for genotyping Geobacillus spp. to provide insight into the prevalence and persistence of certain genotypes within milk powder processing plants.