Permeability barrier of the gram-negative bacterial outer membrane with special reference to nisin

The effect of nisin pretreatment on organic acid-induced permeability increase in strains of Escherichia coli, Pseudomonas aeruginosa, P. marginalis, and Salmonella enterica sv. Typhimurium was investigated, using assays based on the uptake of a fluorescent dye 1-N-phenylnaphthylamine (NPN) and on the bacterial susceptibility to detergent-induced bacteriolysis. The outer membrane of bacteria which had been pretreated with nisin was shown to be less stable against 1 mM EDTA, as indicated by their significantly higher NPN uptake levels as compared to untreated bacteria. Upon challenge with a tenfold lower concentration of EDTA (0.1 mM) some nisin-treated strains (Typhimurium, P. marginalis) exhibited, however, NPN uptake levels which were lower than those seen in control bacteria, suggesting that nisin had stabilized their outer membrane. Nisin pretreatment also decreased the NPN uptake induced by citric or lactic acid or both in E. coli, P. marginalis, and Typhimurium, whereas in P. aeruginosa the pretreatment resulted in increased NPN uptake in response to citric and lactic acid. These results suggest that, with the exception of P. aeruginosa, nisin could protect bacteria from the outer membrane-disrupting effect caused by the acids. P. aeruginosa was, however, shown to be protected against bacteriolysis induced by the detergents sodium dodecylsulfate and Triton X-100. With a pair of isogenic mutants of Typhimurium differing in their cell surface charge it was shown that the NPN uptake response to 1 mM EDTA of the abnormally cationic strain was not significantly affected by nisin, whereas in the normal anionic strain nisin strongly strengthened the uptake. Our hypothesis based on these findings is that the normally anionic cell surface of Gram-negative bacteria has a tendency to bind the cationic nisin. The binding of nisin to the surface does not proceed to the cytoplasmic membrane, but in the outer membrane the bound nisin actually stabilizes its structure through electrostatic interactions. With the exception of EDTA, the organic acids at pH 4 did not cause leakage of cell contents from Typhimurium, indicating that these acids do not permeabilize the outer membrane to an extent required for cytoplasmic pore formation by nisin.     

Chitosan kills bacteria through cell membrane damage

The bactericidal activity of chitosan (CS) acetate solution against Escherichia coli and Staphylococcus aureus was evaluated by the enumeration of viable organisms at different incubation times. Morphologies of bacteria treated with CS were observed by transmission electron microscopy (TEM). The integrity of the cell membranes of both species and the permeabilities of the outer membrane (OM) and inner membrane (IM) of E. coli were investigated by determining the release from cells of materials that absorb at 260 nm, changes in the fluorescence of cells treated with the fluorescent probe 1-N-phenylnaphthylamine (NPN) and release of cytoplasmic beta-galactosidase activity. In addition, the interaction of CS with synthetic phospholipid membranes was studied using gel permeation chromatography (GPC), UV-VIS spectrophotometery, Fourier-transform infrared spectroscopy (FT-IR) and thermal analysis. Results showed that CS increased the permeability of the OM and IM and ultimately disrupted bacterial cell membranes, with the release of cellular contents. This damage was likely caused by the electrostatic interaction between NH(3)(+) groups of CS acetate and phosphoryl groups of phospholipid components of cell membranes.     

Growth and recovery of selected gram-negative bacteria in reconditioned wastewater.

Previous reports indicate that Escherichia coli O157:H7, Salmonella spp., and Vibrio cholerae can grow in nutrient-limited, reconditioned wastewater over the temperature range of 4 to 46 degrees C when the biological oxygen demand of this water is <2, while its coliform growth response (CGR) is >2. In the current study, we investigated the growth response of Vibrio parahaemolyticus, Shigella spp., Vibrio vulnificus, and Pseudomonas aeruginosa in water samples with a CGR of >2 over the temperature range of 4 to 50 degrees C. Both the nonselective media, tryptic soy agar, and the selective media used to identify the pathogen were used for their recovery. The selective media were thiosulfate-citrate-bile-sucrose (TCBS), MacConkey agar (MAC), and Pseudomonas isolation agar (PIA) for the Vibrio, Shigella, and Pseudomonas spp., respectively. V. parahaemolyticus numbers declined rapidly after surviving for 6 days under the nutrient-limiting growth conditions. Shigella spp. did not grow but survived for >28 days at 4 to 25 degrees C. V. vulnificus grew over the narrow temperature range of 12 to 21 degrees C and survived for >21 days at the higher and lower temperature ranges. P. aeruginosa survived and grew during the 14-day test period at 13 to 35 degrees C. Recovery on the nonselective agar gave statistically (P > 0.05) higher numbers than the respective selective media commonly used for these pathogens. These results indicate that caution should be used in attempting direct recoveries using selective media of the four gram-negative bacteria species used in this study from the nutrient-limited water environment.     

Growth of gram-negative bacteria in dry cow secretion

Gram-negative bacteria (n = 192) isolated from infected bovine mammary glands were tested for growth in a pooled source of dry cow secretion. Growth of Klebsiella pneumoniae in dry cow secretion was greater than growth of Escherichia coli and Klebsiella oxytoca. Escherichia coli originating during the early dry period exhibited greater growth in dry cow secretion than those originating around calving or during lactation. Klebsiella pneumoniae growth did not differ with time of origin of intramammary infection. Escherichia coli, K. oxytoca, and K. pneumoniae growth in a synthetic medium was reduced by apolactoferrin plus Ig. Growth reduction was greatest for E. coli. Citrate reversed growth inhibition. The inhibitory properties of dry cow secretion for E. coli may contribute to the low number of naturally occurring intramammary infections originating during the early part of the dry period. Inhibitory properties of dry cow secretion are partially explained by lactoferrin acting in conjunction with antibody to prevent iron acquisition by many gram-negative bacteria.     

壳聚糖的膜性质及其在果蔬保鲜方面的应用研究进展

综述了壳聚糖的成膜性及其在食品保鲜方面的应用研究进展,讨论了壳聚糖的结构包括分子量、脱乙酰化度以及衍生化反应与交联反应对膜性质的影响.壳聚糖膜的组分包括酸的种类和浓度、增塑剂的种类和浓度与其他高分子材料复合等对膜性质的影响.介绍了壳聚糖对果蔬的涂膜保鲜效果.本文对开发壳聚糖在果蔬防腐保鲜方面的应用具有理论指导意义.     

A study on the prevalence of gram-negative bacteria in bulk tank milk

Bulk tank milk from 131 dairy herds in eastern South Dakota and western Minnesota were examined for coliforms and noncoliform bacteria. Coliforms were detected in 62.3% of bulk tank milk samples. Counts ranged from 0 to 4.7 log10 cfu/ml. The mean count was 3.4 log10 cfu/ml. Gram-negative noncoliform bacteria were observed in 76.3% of bulk tank milk. Counts ranged from 0 to 6.2 log10 cfu/ml. The mean count was 4.8 log10 cfu/ml. A total of 234 isolates from bulk tank milk were examined to species level; 205 isolates belonged to 28 species. Coliforms and gram-negative noncoliform bacteria accounted for 32.9 and 67.1% of the total isolates, respectively. Organisms such as Agrobacterium radiobacter, Bordetella spp., Comamonas testosteroni, Listonella damsela, Ochrobactrum anthropi, and Oligella urethralis were isolated from bulk tank milk in this study. These organisms have not been reported previously in bulk tank milk. A total of 116 isolates of Pseudomonas spp. were isolated from raw milk; 98 isolates belonged to nine Pseudomonas spp., and the remaining 18 isolates could not be identified to their species level. Pseudomonas was the most predominant genus. Pseudomonas fluorescens was the most predominant species isolated from bulk tank milk and accounted for 29.9% of all isolates examined. The results of the study suggest that counts of coliforms and noncoliform bacteria in bulk tank milk vary considerably. The isolates represent a wide variety of Gram-negative bacterial species. Examination of bulk tank milk for coliforms and noncoliform bacteria could provide an indication of current and potential problems associated with bacterial counts and milk quality.     

Antimicrobial resistance of gram-negative enteric bacteria from pigs in a nonantimicrobial-exposed herd before and after transportation.

Loading pigs onto trucks and transporting them for 30 min resulted in a significant increase in proportion of antimicrobial resistance of gram-negative enteric bacteria in fecal material. Similarly, the mean number of antimicrobial agents in the resistance patterns of these bacteria increased during loading and transportation. However, the increases were of a transient nature, as resistance values were similar to those of a nontransported control group 1 day after the pigs had been transported.     

Sensitization of gram-negative and gram-positive bacteria to jenseniin G by sublethal injury

Jenseniin G, a bacteriocin produced by Propionibacterium thoenii P126, is active against related propionibacteria and some lactic acid bacteria and is sporostatic to botulinal spores. The objective of this study was to evaluate the effects of sublethal stress on jenseniin G activity. Bacillus cereus, Enterococcus faecalis, Escherichia coli, Listeria monocytogenes, Salmonella Typhimurium, Shigella flexneri, Staphylococcus aureus, and Yersinia enterocolitica were subjected to temperature, acid, and osmotic stresses in the presence of jenseniin G. The bacteriocin reduced the viability of sublethally injured cultures, although the extent of reduction varied with strain and treatment. E. faecalis was the most sensitive to temperature stress; no reduction of viable counts occurred in the absence of jenseniin G, and a 1.5-log reduction occurred in the presence of jenseniin G. B. cereus, L. monocytogenes, and S. aureus were more sensitive to jenseniin G when exposed to heat stress than when exposed to cold stress, whereas E. coli, Salmonella Typhimurium, and S. flexneri were more sensitive to jenseniin G when exposed to cold stress than when exposed to heat stress. When comparing an acid stress test alone to a combination of acid stress and jenseniin G, E. faecalis and L. monocytogenes showed the greatest sensitivities with 4.87- and 2.82-log reductions, respectively, after 7 days. All cultures except for S. aureus were adversely affected by the combination of salt stress and jenseniin G. Salmonella Typhimurium showed the greatest sensitivity to salt stress with jenseniin G (a 1.54-log reduction at day 7) when compared to salt stress alone (a 0.55-log reduction at day 7). Jenseniin G, like bacteriocins produced by other gram-positive species, has broader activity against stressed organisms.     

Assessing the ability of nisin A and derivatives thereof to inhibit gram-negative bacteria from the genus Thermus

Nisin is a bacteriocin that is globally employed as a biopreservative in food systems to control gram-positive, and some gram-negative, bacteria. Here we tested the bioactivity of nisin A-producing Lactococcus lactis NZ9700 and producers of bioengineered variants thereof against representatives of the gram-negative genus Thermus, which has been associated with the pink discoloration defect in cheese. Starting with a total of 73 nisin variant-producing Lactococcus lactis, bioactivity against Thermus was assessed via agar diffusion assays, and 22 variants were found to have bioactivity greater than or equal to that of the nisin A-producing control. To determine to what extent this enhanced bioactivity was attributable to an increase in specific activity, minimum inhibitory concentrations were determined using the corresponding purified form of these 22 nisin A derivatives. From these experiments, nisin M17Q and M21F were identified as peptides with enhanced antimicrobial activity against the majority of Thermus target strains tested. In addition, several other peptide variants were found to exhibit enhanced specific activity against a subset of strains.     

Survival and detection of coliforms,Enterobacteriaceae, and gram-negative bacteria in Greek yogurt

Despite the widespread use of coliforms as indicator bacteria, increasing evidence suggests that the Enterobacteriaceae (EB) and total gram-negative groups more accurately reflect the hygienic status of high-temperature, short-time pasteurized milk and processing environments. If introduced into milk as postpasteurization contamination, these bacteria may grow to high levels and produce a wide range of sensory-related defects. However, limited information is available on the use and survival of bacterial hygiene indicators in dairy products outside of pasteurized fluid milk and cheese. The goal of this study was to (1) provide information on the survival of a diverse set of bacterial hygiene indicators in the low pH environment of Greek yogurt, (2) compare traditional and alternative detection methods for their ability to detect bacterial hygiene indicators in Greek yogurt, and (3) offer insight into optimal hygiene indicator groups for use in low-pH fermented dairy products. To this end, we screened 64 bacterial isolates, representing 24 dairy-relevant genera, for survival and detection in Greek yogurt using 5 testing methods. Before testing, isolates were inoculated into plain, 0% fat Greek yogurt (pH 4.35 to 4.65), followed by a 12-h hold period at 4 ± 1°C. Yogurts were subsequently tested using Coliform Petrifilm (3M, St. Paul, MN) to detect coliforms; Enterobacteriaceae Petrifilm (3M), violet red bile glucose agar and the D-Count (bioMérieux, Marcy-l'Étoile, France) to detect EB; and crystal violet tetrazolium agar (CVTA) to detect total gram-negative bacteria. Overall, the non-EB gram-negative isolates showed significantly larger log reductions 12 h after inoculation into Greek yogurt (based on bacterial numbers recovered on CVTA) compared with the coliform and noncoliform EB isolates tested. The methods evaluated varied in their ability to detect different microbial hygiene indicators in Greek yogurt. Crystal violet tetrazolium agar detected the highest portion of coliforms, whereas EB Petrifilm detected the highest portion of EB, as well as highest portion of total gram-negative bacteria. Additionally, the D-Count method allowed for faster detection of EB in yogurt by generating results in approximately 13 h rather than the 24 h required when using EB Petrifilm and violet red bile glucose agar. Results from this study indicate that the coliform and EB groups encompass a broad range of dairy-relevant gram-negative bacteria with the ability to survive in Greek yogurt, supporting their use as microbial hygiene indicator groups in low-pH fermented dairy products.     

壳聚糖对冷却猪肉品质特性的影响研究

对壳聚糖在贮藏期间对冷却猪肉的感官性状(色泽、持水力)、理化品质(pH值、TBA、TVB-N)和微生物品质(菌落总数)的影响进行研究。添加壳聚糖能够明显的抑制微生物的生长(p<0.05),随着壳聚糖添加浓度的增大,脂质氧化率、挥发性盐基氮增加率及pH值上升率均下降,而持水力则呈现上升趋势。经过1.5%壳聚糖处理的样品在各方面的综合品质最好。     

Autoinducer-2 activity of gram-negative foodborne pathogenic bacteria and its influence on biofilm formation

ABSTRACT:  This study evaluated whether autoinducer-2 (AI-2) activity would be associated with biofilm formation by Salmonella and Escherichia coli O157:H7 strains on food contact surfaces. In study I, a Salmonella Typhimurium DT104 strain and an E . coli O157:H7 strain, both AI-2 positive, were individually inoculated into 50 mL of Luria–Bertani (LB) or LB + 0.5% glucose (LBG) broth, without or with stainless steel or polypropylene ( Salmonella ) coupons. At 0, 14 ( Salmonella ), 24, 48, and 72 h of storage (25 °C), cells in suspension and detached cells from the coupons, obtained by vortexing, were enumerated on tryptic soy agar. In study II, a Salmonella Thompson AI-2-positive strain and an AI-2-negative strain, and an E . coli O157:H7 AI-2-positive strain and an AI-2-negative strain were inoculated into LB broth with stainless steel coupons. Cells were enumerated as in study I. In both studies, AI-2 activity was determined in cell-free supernatants. Cell numbers of S . Typhimurium DT104 on biofilms were higher ( P < 0.05) in LB than those in LBG, while the E . coli O157:H7 strain showed no difference ( P ≥ 0.05) in biofilm cell counts between LB and LBG after storage for 72 h. Both S . Typhimurium DT104 and E . coli O157:H7 strains produced higher ( P < 0.05) AI-2 activity in LBG than LB cell suspensions. Cell counts of AI-2-positive and-negative S . Thompson and E . coli O157:H7 strains were not different ( P ≥ 0.05) within suspensions or coupons (study II). The results indicated that, under the conditions of this study, AI-2 activity of the pathogen strains tested may not have a major influence on biofilm formation on food contact surfaces, which was similar between AI-2-positive and -negative strains.     

壳聚糖/卡拉胶聚电解质复合物的制备及其性能的基础研究

用天然多糖壳聚糖和卡拉胶合成壳聚糖/卡拉胶聚电解质复合物(PEC),探讨了PEC合成过程中的反应机理,考察PEC聚电解质在不同pH缓冲溶液中的稳定性和做药物释放载体的可能性。     

益生菌功能特性研究

采用牛津杯法测定益生菌PB1，A878，M1，PB2，A30，A31对肠道致病菌的抑制性；接种于高胆固醇培养基中，37℃厌氧培养后测定菌株降解胆固醇的量；将株Pba，PB1，M1，A30每天以0．5mL／只的量灌胃给正常小白鼠，30d后测定小鼠免疫器官指数和血清中细胞因子的变化。结果表明，PB1，A878和Pba具有较强的抑菌能力；益生菌降解胆固醇的量为38％～52％；所测益生菌均能提高小鼠血清中IL-2，IL-6和TNF-α的含量。本研究的结论为益生菌功能食品的开发提供理论依据。     

Evaluation of PCR-single-strand conformational polymorphism analysis for identification of gram-negative histamine-producing bacteria isolated from fish

In this study, the previously established PCR-single-strand conformation polymorphism (SSCP) system for detecting and identifying gram-negative histamine-producing bacteria was evaluated. This system can detect and identify histamine-producing bacteria directly from seafood by the use of sequence polymorphisms of the histidine decarboxylase gene (hdc). First, we isolated 81 histamine-producing strains of bacteria from fish samples and analyzed the hdc gene by the PCR-SSCP system. The 22 newly obtained SSCP banding patterns were added to our database, and the utility of our modified database was tested in a second experiment consisting of 18 strains of histamine-producing bacteria isolated from 25 fish samples. Approximately 80% of the histamine-producing strains corresponded to those in the new database. Use of the database for PCR-SSCP analysis, including the band patterns newly added in this study, for the hdc gene makes it possible to more accurately identify histamine producers.     

Enhancing the barrier properties of colloidosomes using silica nanoparticle aggregates

Recent interest is focused on microcapsules stabilized using colloidal nanoparticles – termed ‘colloidosomes – for encapsulation applications in food, drug and cosmetic industries. However, due to electrostatic repulsion between similarly charged particles, shells composed of single-type nanoparticles tend to be monolayer-thick and relatively permeable. We investigated a self-assembly method for controlling the permeability of colloidal shells using aggregates composed of oppositely charged silica nanoparticles. Using a combination of rapid fluorescence based method and theoretical diffusion models, we found that colloidosomes whose shells contained colloidal silica aggregates displayed lower permeability to peroxyl radicals than ones stabilized by single type of silica nanoparticles. Furthermore, the permeability varied as a function of the ratio of oppositely charged silica nanoparticles in the shell. The ability to control the permeability of colloidosomes, while using a simple self-assembly synthesis method, will enable enhanced control over release kinetics and oxidative stability of encapsulants.     

Conventional tube and microplate Limulus amoebocyte lysate procedures for determination of gram-negative bacteria in milk

A comparison was made of the conventional tube and microplate Limulus amoebocyte lysate assay for detection of gram-negative bacterial lipopolysaccharide in milk. Raw whole milk samples were maintained frozen and portions were examined periodically on alternate days during 13-d storage to evaluate the reproducibility of both Limulus amoebocyte lysate procedures and to determine optimum reaction conditions for the microplate method. One-day-old, raw and locally purchased pasteurized milk samples, held at 7 degrees C, were analyzed during storage to establish the correlation of both procedures with aerobic and modified psychrotrophic plate counts. Vitamin- and mineral-fortified dairy-based products were examined using the microplate Limulus amoebocyte lysate test as a potential indicator of raw material or finished product bacterial quality and possible postprocessing contamination. Statistical analysis of the data collected comparing the conventional tube and the microplate Limulus amoebocyte lysate assay demonstrated no significant difference exists between the methods when either the modified psychrotrophic bacterial count or the aerobic plate count was used to determine gram-negative bacteria in pasteurized or raw milk (P less than .91). The microplate method, which uses half the lysate reagent, was a good indicator of the bacterial quality of milk and fortified dairy products, consistently detecting bacterial levels greater than 10(3) to 10(4)/ml.     

Porosity and barrier properties of polyethylene meltblown nonwovens

There are several areas in which textiles have been highly desirable due to their specific and high performance for many industries and sectors including in civil engineering, automobile, medicine, filtration, and electronics. Polymer used has a significant effect on the properties of meltblown nonwovens. Polypropylene being the most commonly used polymer, very limited data have been reported on the polyethylene meltblown nonwovens which are desired due to their unique processing and performance advantages. In this study, we have investigated the effects of process variables, die temperature, air pressure, and die-to-collector distance on some characteristics of polyethylene meltblown nonwovens such as pore size, air permeability, hydrostatic head, and SEM analysis. It was observed that the variables have significant effects on the produced nonwovens, and many of the properties are comparable to those obtained with that of polypropylene meltblown webs.