Attachment and biofilm formation by various serotypes of Salmonella as influenced by cellulose production and thin aggregative fimbriae biosynthesis

This study was undertaken to quantify thin aggregative fimbriae and cellulose produced by Salmonella and to evaluate their roles in attachment and biofilm formation on polystyrene and glass surfaces. Thin aggregative fimbriae and cellulose produced by four wild-type and two pairs of Salmonella, representing four different colony morphotypes (rdar: red, dry, and rough; pdar: pink, dry, and rough; bdar: brown, dry, and rough; and saw: smooth and white), were quantified. The ability of the Salmonella cells to attach and form biofilms on the selected surfaces was evaluated in Luria-Bertani (LB) broth with or without salt (0.5%) or glucose (2%) at 28 degrees C during a 7-day period. The cells expressing the rdar or pdar colony morphotypes produced significantly greater amounts of thin aggregative fimbriae and cellulose on LB no salt agar, respectively. The cells expressing the rdar colony morphotype attached in higher numbers and formed more biofilm than did the cells expressing the pdar colony morphotype. The members of the pairs expressing the bdar colony morphotype attached more efficiently and formed more biofilm on the tested surfaces than did their counterparts expressing the saw colony morphotype. These results indicated that thin aggregative fimbriae impart attachment ability to Salmonella and, upon coexpression with cellulose, enhance biofilm formation on certain abiotic surfaces. The knowledge acquired in the study may help develop better cleaning strategies for food processing equipment.     

Biofilm formation and cell surface properties of Staphylococcus aureus isolates from various sources

Food Science and Biotechnology - This study investigated biofilm formation, cell surface hydrophobicity, colony spreading, and slime production for 112 Staphylococcus aureus strains isolated from...     

Biofilm formation and the survival of Salmonella Typhimurium on parsley

Although several studies provide evidence that the formation of biofilms by human pathogens on plant tissue is possible, to date there is no direct evidence that biofilms enhance the resistance of plant-associated pathogens to disinfectants or biocides. We hypothesized that biofilm formation would enhance the adhesion and survival of Salmonella on leafy vegetables. To test our hypothesis, we compared the adhesion and persistence of Salmonella Typhimurium and its biofilm-deficient isogenic mutant. Following inoculation of parsley and rinsing with water or chlorine solution, both strains had similar survival properties, and up to 3-log reduction were observed, depending on chlorine concentration. This indicates that the biofilm matrix of Salmonella likely does not play a significant role in initial adhesion and survival after disinfection. After a week of storage the biofilm producing strain survived chlorination significantly better than the biofilm-deficient mutant. However, the recovery of the mutant was still elevated, indicating that although the biofilm matrix has a role in persistence of Salmonella after chlorination treatment of parsley, this is not the most important mechanism, and other mechanisms, probably the ability to penetrate the plant tissue or the pre-existing biofilms, or production of different polysaccharides other than cellulose, provide the protection.     

Biofilm formation,virulence gene and multi-drug resistance in Salmonella Kentucky isolated in Tunisia

Food-borne diseases caused by Salmonella enterica are a significant public health concern around the world. Since 2002, S. enterica serovar Kentucky has shown an increase in several countries with the concurrent emergence of multidrug-resistant isolates. The spread of such strains in the environment poses a major public health problem. A total of 57 Salmonella Kentucky strains isolated from different sources during the period 2005 to 2008 in Tunisia, were characterized by their antimicrobial and mercury resistance profiles; ability to form a biofilm; virulence invA/spvC genes and quorum sensing sdiA gene. A total of 10.6% of the isolates demonstrated multidrug-resistance against 3 to 13 antibiotics with ciprofloxacin resistance occurring in 33% of human isolates. In addition, 37% of the isolates exhibited minimum inhibitory concentrations value to mercuric chloride, ranging from 8 to 32 μg ml^?1 and were considered as resistant strains. The majority of strains tested were able to form a biofilm, especially for environmental and animal derived isolates. Therefore, the biofilm seems to comprise a normal and favorable capability in the life of Salmonella Kentucky in the environment. Interestingly, all the isolates possessed the sdiA gene, 87.7% of isolates possessed the invA gene, and no isolate harbored the spvC gene.The emergence of resistance to ciprofloxacin in human Salmonella Kentucky isolates, added to the presence of invA and sdiA genes, and the production of biofilm could be the decisive factors in the dissemination of S. Kentucky strains on a large scale.     

Biofilm formation by multidrug-resistant Salmonella enterica serotype typhimurium phage type DT104 and other pathogens

The biofilm-forming capability of Salmonella enterica serotypes Typhimurium and Heidelberg, Pseudomonas aeruginosa, Listeria monocytogenes, Escherichia coli O157:H7, Klebsiella pneumoniae, and Acinetobacter baumannii isolated from humans, animal farms, and retail meat products was evaluated by using a microplate assay. The tested bacterial species showed interstrain variation in their capabilities to form biofilms. Strong biofilm-forming strains of S. enterica serotypes, E. coli O157: H7, P. aeruginosa, K. pneumoniae, and A. baumannii were resistant to at least four of the tested antibiotics. To understand their potential in forming biofilms in food-processing environments, the strong biofilm formers grown in beef, turkey, and lettuce broths were further investigated on stainless steel and glass surfaces. Among the tested strains, Salmonella Typhimurium phage type DT104 (Salmonella Typhimurium DT104) isolated from retail beef formed the strongest biofilm on stainless steel and glass in beef and turkey broths. K. pneumoniae, L. monocytogenes, and P. aeruginosa were also able to form strong biofilms on the tested surface materials. Salmonella Typhimurium DT104 developed a biofilm on stainless steel in beef and turkey broths through (i) initial attachment to the surface, (ii) formation of microcolonies, and (iii) biofilm maturation. These findings indicated that Salmonella Typhimurium DT104 alongwith other bacterial pathogens could be a source of cross-contamination during handling and processing of food.     

Antibiotic resistance of Salmonella spp. from animal sources in Spain in 1996 and 2000

Emergence of resistant and multiresistant bacteria has become an important worldwide sanitary problem. International agencies recommend improving resistance surveillance studies in not only human but also animal origin strains. Because of its ubiquitous characteristics and zoonotic agent consideration, Salmonella spp. can be used as a good indicator microorganism for resistance surveillance studies. Salmonella spp. strains from animal sources isolated in 1996 (107) and 2000 (474) in Spain were tested against 12 different antimicrobials agents, using the disc diffusion method. Results were interpreted following the NCCLS criteria. Data showed that Salmonella spp. strains (61.7% in 1996 and 81.5% in 2000) were resistant to at least one antibiotic. Pig-related strains were considerably more resistant than strains from other sources. Enteritidis serotype was less resistant than other serotypes, except for ampicillin in 1996 (50% resistant) and nalidixic acid in 2000 (65.1% resistant). An emergent monophasic serotype, 4,5,12:i:-, first detected in 1997 in Spain was 100% resistant and 90% multiresistant. Typhimurium serotype was the most common Salmonella serotype from animal sources in both years. It was widely distributed among animals and was among the serotypes with a higher degree of resistance. The ampicillin, chloramphenicol, sulfonamides, streptomycin, and tetracycline resistance pattern, commonly associated with Salmonella serotype Typhimurium DT 104, had spread among other Typhimurium phage types and other Salmonella serotypes. Salmonella spp. strains isolated from feeding stuffs were considerably more susceptible than animal source strains, suggesting that the high Salmonella spp. resistance percentage was probably due to the use of antibiotics in animal farms rather than the consumption of contaminated feeding stuffs.     

肉蛋类食品沙门氏菌分离与菌膜检测

从天津市市内6区和塘沽区各农贸市场购买肉蛋类样品共计78份,利用国标方法从中分离沙门氏菌,根据种内特异性基因片段设计3对引物利用PCR方法对分离菌株进行鉴定,结果共鉴定沙门氏菌4株;利用结晶紫染色方法对4株沙门氏菌菌膜形成能力进行鉴定,其中2株菌膜形成能力较强。该试验结果能够有效反映天津市内肉蛋类食品卫生状况,并对沙门氏菌的控制提供有益参考。     

Effects of carbon and nitrogen sources, sodium chloride and culture conditions on cytotoxin production by Salmonella choleraesuis

Factors governing Bacillus cereus colony growth on agar media as modified by the agar content (1-7%, w/v) were studied. Agar had a significant effect on the radial growth rate which diminished as the agar content increased. Cell density in colonies (colony density) was found to decrease during the incubation time, with lower values occurring in the presence of 1% agar. Size and DNA content of the cells grown on 1 and 7% agar were similar. An increasing proportion of cell population growing on 7% agar produced spores during the 24-h incubation period. It was shown that 'water condition' on the agar surface could be associated with colony density, with 7% agar media presenting the thinnest nominal thickness of the liquid film. On the other hand, the partial drying phenomena of the agar media which occur during preparation and incubation, could not account for the observed differences in colony growth.     

PCR identification of Salmonella: potential contamination sources from production and postharvest handling of cantaloupes

Salmonella is one of the most frequently reported etiological agents in outbreaks of foodborne diseases associated with the consumption of cantaloupes. Sensitive and reliable methods for detecting and identifying foodborne microorganisms are needed. The PCR can be used to amplify specific DNA fragments and thus to detect and identify pathogenic bacteria. In this study, a PCR method was used to evaluate the incidence of Salmonella at cantaloupe production, harvest, and packaging steps, and the results were compared with those of the standard method for detection of Salmonella in foods (Mexican NOM-114-SSA1-1994). Salmonella was detected by both standard and PCR methods in 23.5% of the irrigation water samples but only by the PCR method in 9.1% of the groundwater samples, 4.8% of the chlorinated water samples, 16.7% of samples from the hands of packing workers, 20.6% of samples from the packed cantaloupes, and 25.7% of samples from the in-field cantaloupes. With the standard method, Salmonella was found in 8.3% of the crop soil samples. Statistical analysis indicated a significant difference in sensitivity (P < 0.05) between the two methods; the PCR method was 4.3 times more sensitive than the standard method. Salmonella was found at seven of the eight pointsevaluated during the production and postharvest handling of cantaloupe melons.     

Genetic diversity and virulence gene determinants of antibiotic-resistant Salmonella isolated from preharvest turkey production sources

This study evaluated the molecular diversity of 29 Salmonella serotypes isolated from turkey ceca and the production environment. Isolates were resistant to bacitracin (100%), erythromycin (100%), novobiocin (100%), rifampin (100%), streptomycin (62%), gentamicin (52%), spectinomycin (48%), tetracycline (31%), sulfamethoxazole/trimethoprim (SXT) (3%) and tobramycin (3%). The minimum inhibitory concentration (MIC) values ranged from 32 to >/=1024 microg/ml. The pulsed-field gel electrophoresis (PFGE) and ribotyping patterns were identical within each of the serotypes Heidelberg, Worthington and Muenster. The plasmid profiles were identical within each of the Salmonella serotypes. Two different clones of Salmonella anatum were differentiated by PFGE typing but not by ribotyping. Heidelberg isolates from nine turkey ceca and three drinker samples had identical antibiotic resistance, PFGE, ribotype and plasmid patterns, suggesting that transmission of this particular clone may have occurred between the birds and the drinkers. Identical PFGE, ribotype and plasmid patterns were observed in one Salmonella worthington isolate from turkey ceca in one flock and two S. worthington isolates from feeder contents and drinkers from a subsequent flock, suggesting transmission of this pathogen between flocks. Individual and multiple polymerase chain reaction (PCR) analyses revealed the presence of the virulence genes invA, aceK and sopB and the absence of the h-1i gene in all isolates. A combination of genotypic and phenotypic markers can be useful in studying genetic variation among natural salmonellae populations in turkey production and delineating possible transmission pathways.     

In vitro availability of calcium from sources of cellulose,methylcellulose, and psyllium

Certain types of dietary fibres may decrease the amount of dietary calcium available for absorption. The objective of this study was to determine the in vitro calcium availability from sources of cellulose, methylcellulose, and psyllium, such as cereals, fibre supplements, and purified fibres. Total dietary fibre, soluble and insoluble dietary fibre, acid detergent fibre, neutral detergent fibre, cellulose, hemicellulose, lignin, protein, uronic acid, phytate, water holding capacity, and total endogenous calcium content were determined. Results from the chemical analyses of the fibre sources varied significantly. Free endogenous calcium, at pH 3–8, was determined via gel filtration. Calcium was added to each fibre source and free exogenous calcium was determined via gel filtration at pH 1 and pH 8. The analysis of variance procedure was used to determine differences in binding capacities of the fibres. There was virtually no binding of exogenous calcium by sources of cellulose, methylcellulose, or psyllium.     

Biofilm formation by lactobacilli and resistance to stress treatments

We investigated biofilm formation by six lactobacilli isolates and tested the resistance of biofilm and planktonic Lactobacillus plantarum B1 and Lactobacillus paracasei W1 to ethanol (ET) and acetic acid (AA). The viability of both isolates was considerably affected by ET, with a higher reduction observed in the planktonic cells, whereas AA was harmful only to L. paracasei W1. Cells that had intact (Syto^® 9⁺PI⁻) and compromised (Syto^® 9⁻PI⁺) membranes, and adopted intermediate states with different concentrations of the probes, i.e., Syto^® 9⁺PI⁺ and Syto^® 9⁻PI⁻, were identified following flow cytometry. We linked Syto^® 9⁺PI⁺ to the planktonic cells and clarified on Syto^® 9⁻PI⁻ cells, which after being sorted could regain growth better in biofilm cells. Based on confocal laser scanning microscopy, numerous cells had damaged membranes in the ET-treated biofilm, while half the cells in the AA-treated biofilm maintained their membrane integrity.     

Biofilm formation and exopolysaccharide (EPS) production by Cronobacter sakazakii depending on environmental conditions

Biofilm matrices are formed largely of extracellular polymeric substance (EPS). This study was conducted to investigate biofilm formation and EPS production by Cronobacter sakazakii under various conditions (media, nutrition, and relative humidity (RH)) by quantification of EPS and cell populations, Field Emission Scanning Electron Microscope (FE-SEM), and colony observation. Various agar media conditions (TSA without dextrose (W/D), M9 minimum salt medium (MSM) agar, and M9 MSM agar with 3% glucose, 3% NaCl, 3% Tween 80, 3% sucrose, and adjusted to pH 5 with HCl) were prepared. C. sakazakii biofilm formed on the surface of stainless steel coupons (SSCs) immersed in TSB W/D and M9 MSM with or without 0, 1, 3, and 5% sucrose and subsequently exposed to various RH levels (23, 43, 68, 85, and 100%). EPS production by C. sakazakii on TSA W/D was significantly higher than that on other media after 1 and 2 days. However, C. sakazakii ATCC 12868 produced the highest levels of EPS (209.18 ± 16.13 and 207.22 ± 4.14 μg/mL after 1 and 2 days, respectively) on M9 MSM agar with 3% sucrose. Regarding C. sakazakii ATCC 12868 biofilm formed on the surface of SSCs immersed in M9 MSM with 0, 1, 3, and 5% sucrose and subsequently exposed to various RHs, populations were significantly different among the various RHs and sucrose concentrations, and EPS production was significantly higher (4.69 mg/L) compared to other sucrose concentrations (0%:0.71 mg/L and 1%:0.98 mg/L), except for M9 MSM with 3% sucrose (2.97 mg/L) (P ≤ 0.05). From these results, biofilm formation and EPS production by C. sakazakii differed depending on the nutrient or environmental conditions provided to the cells.     

利用SSF制取纤维乙醇的工艺研究

利用同步糖化发酵（SSF）技术,以汽爆玉米秸秆为主要原料,对纤维乙醇的发酵工艺进行研究。玉米秸秆经蒸汽爆破预处理后,酶解得率增大到85.0%。进一步利用Box-Behnken实验设计方法,选取酶用量、发酵温度和发酵时间为影响乙醇产率的主要因素,通过响应面分析得到了较优的工艺条件：底物浓度15%（w/v）,酶用量35FPU/g（底物）,发酵温度37℃,发酵时间90h。在优化的工艺条件下,乙醇浓度为42.2g/L,达到理论产量的82.6%。和分步糖化发酵（SHF）工艺结果比较,SSF具有更高的生产效率。     

Biofilm formation, extracellular polysaccharide production, and cell-to-cell signaling in various Enterobacter sakazakii strains: aspects promoting environmental persistence

Enterobacter sakazakii is considered an opportunistic pathogen and has been implicated in food-associated cases of meningitis or enteritis, especially in neonates and infants. The organism has been detected in various types of food and in food production units, but so far only powdered infant formula has been linked to outbreaks of disease. Survival and persistence in such environments requires the ability to adapt to high osmotic potentials and/or dry conditions. Fifty-six E. sakazakii strains were evaluated for several features important for persistence and survival: (i) biofilm formation and the putative production of cellulose as one of the components of the extracellular matrix, (ii) adherence to hydrophilic and hydrophobic surfaces, (iii) the production of extracellular polysaccharides, and (iv) the ability of E. sakazakii to produce cell-to-cell signaling molecules. Pellicle and flock formation was observed in 21 of the strains grown in Luria-Bertani broth and in 44 of the strains grown in brain heart infusion broth. Calcofluor-stained fibrils, observed microscopically in every (fragile or rigid) pellicle, suggested the presence of cellulose as an extracellular compound in this type of biofilm. Twelve isolates did not form any pellicle or flocks under either condition. Twenty-three of the isolates exhibited the potential to adhere to glass surfaces in shaken cultures, and 33 strains showed biofilm formation at the air-solid interface of polyvinyl chloride microtiter wells. Sixteen isolates adhered to both surfaces. Twenty-four of the isolates tested produced a milky, viscous mass, considered as extracellular polysaccharide. High-performance liquid chromatography analysis of the polysaccharide revealed the presence of glucose, galactose, fucose, and glucuronic acid. Thin-layer chromatography analyses performed on ethyl acetate extracts of cell-free supernatants of the 56 strains indicated the presence of two different types of acylated homoserine lactones (3-oxo-C6-HSL and 3-oxo-C8-HSL). These findings illustrate the ability of E. sakazakii to produce cell-to-cell signaling molecules.     

Biofilm formation by Staphylococcus aureus strains and their control by selected phytochemicals

Biofilm formation by 30 Staphylococcus aureus dairy isolates and their control by phytochemicals were investigated. The majority of strains were biofilm positive by phenotypic analysis. The nuc and icaA genes were present in 30 and 27 strains, respectively. In addition, 13 strains were positive for all nuc, clfA, fnbA and icaA genes. The antimicrobial and antibiofilm activities of citral, cinnamaldehyde, eugenol, farnesol, limonene and terpinen‐4‐ol were also evaluated for seven strains. It was shown that the use of farnesol, cinnamaldehyde or terpinen‐4‐ol at a concentration of 2 mg/mL could be at least 80% effective on S. aureus strains and their biofilms.     

Location and limitation of cellulose production by Acetobacter xylinum established from oxygen profiles

The static fermentation of coconut water sucrose by Acetobacter xylinum was carried out at initial pH's of 3.0, 4.0, 5.0 or 6.0. Cellulose was produced at the surface, and its production was most favourable at pH's 4.0 and 5.0. These pH values also allowed for optimal bacterial growth. Oxygen concentration profiles were measured with microelectrodes at different cultivation stages, and steep profiles were obtained with penetration depths between 50 and 100 microm. A substrate penetration depth analysis confirmed the hypothesis that the first stage of the fermentation is entirely oxygen controlled. Diffusion calculations showed, however, that at a later stage sucrose becomes a limiting substrate also, which was confirmed by the decrease in cellulose production rate over time. The effective diffusion coefficient of oxygen in deactivated cellulose pellicles was measured with microelectrodes, and a value of 1.4 x 10(-9) m2/s was obtained under all investigated conditions. The oxygen flux was 5.9 x 10(-6) mol/m2.s, while a significantly higher value of 9.1 x 10(-6) mol/m2.s was obtained at pH 4.0.     

Reduction of Salmonella Typhimurium and Listeria monocytogenes on produce by trisodium phosphate

The application of trisodium phosphate (TSP) on produce against food-borne bacteria has not been extensively evaluated. This research studied the effect of 20 and 50 mg/ml TSP in reducing Salmonella Typhimurium (ST) and Listeria monocytogenes (Lm) on model produce (lettuce and peppers). Washed and air-dried Iceberg lettuce (3 × 3 cm²) and Jalapeno peppers (25–30 g) were spiked with S. Typhimurium or L. monocytogenes (～7 log₁₀ CFU/ml). Samples were treated with 20 or 50 mg/ml TSP, 200 mg/L sodium hypochlorite (for comparison with traditional washes) or water (control rinse) for 15 or 30 s. Treatments were immediately neutralized with trypticase soy broth (TSB) containing 30 mg/ml beef extract, serially diluted, plated on Xylose Lysine Tergitol 4 (XLT4) agar for ST and Tryptic Soy Agar (TSA) for Lm and incubated at 37 °C for 24–48 h. Results showed that 20 mg/ml TSP and 200 mg/L sodium hypochlorite caused 5–6 log₁₀ CFU/ml reduction in ST on both lettuce and peppers after 15 s and 30 s, while 50 mg/ml TSP reduced ST to undetectable levels on both produce at both contact times. For overnight Lm cultures, a mere reduction of 0.21 and 0.28 log₁₀ CFU/ml was obtained using 20 and 50 mg/ml TSP after even 5 min, while 200 mg/L sodium hypochlorite decreased Lm by ～1 log₁₀ CFU/ml after 30 s and 1 min on lettuce, and decreased pure culture Lm to undetectable levels after 3 min. Thus, 50 mg/ml TSP appears effective against ST with negligible effects against Lm, whose mode of action needs to be understood.     

近红外技术对不同动物来源肉掺假的检测

采用近红外光谱结合主成分分析法(PCA)、判别分析法,分别建立了牛肉和羊肉中掺杂其它动物肉的定性鉴别模型,根据鉴别准确率评价模型的预测性能。采用近红外光谱结合PCA、偏最小二乘法(PLS),建立了掺假物的定量检测模型,根据模型对预测集样品的预测均方差(RMSEP)以及预测值与实测值间的相关系数(r)验证模型的预测能力。结果,牛肉掺猪肉模型对训练集和预测集的鉴别准确率分别为97.86%和91.23%,羊肉掺猪肉模型对训练集和预测集的鉴别准确率分别为98.28%和92.98%,羊肉掺鸭肉模型对训练集和预测集的鉴别准确率分别为99.59%和93.97%,羊肉掺假模型对训练集和预测集的鉴别准确率分别为97.57%和90.76%。牛肉掺假定量模型对训练集的交互验证均方差(RMSECV)和预测集的RMSEP分别为3.87%和4.13%,r分别为0.9505和0.9134;羊肉掺假定量模型对训练集的RMSECV和预测集的RMSEP分别为4.48%和4.86%,r分别为0.9306和0.9082。表明近红外技术结合一定的化学计量学方法可实现不同动物来源肉掺假的鉴别,且能够对掺假物进行定量检测。