Detection and characterization of virulence genes and integrons in Aeromonas veronii isolated from catfish

The presence of virulence genes and integrons was determined in 81 strains of Aeromonas veronii isolated from farm-raised catfish. Polymerase chain reaction (PCR) protocols were used to determine the presence of genes for cytotoxic enterotoxin (act), aerolysin (aerA), two cytotonic enterotoxins (ast, alt), lipase (lip), glycerophospholipid:cholesterol acyltransferase (gcaT), serine protease (ser), DNases (exu), elastase (ahyB) and the structural gene flagellin (fla) in the template DNA. Oligonucleotide primers amplified a 231-bp region of the act gene from the template DNA of 97.0% of the isolates. Primers specific for the amplification of the aerA gene amplified a 431-bp region of the aerA gene from the template DNA of 96.0% of the isolates. None of the isolates contained ast or alt genes. Oligonucleotide primers specific for the amplification of lip, gcaT, ser and fla genes, amplified their respective amplicons from 85.0, 78.0, 82.0 and 80.0% of the isolates. None of the isolates contained exu or the elastase genes. Several of the isolates (48.0%) contained class I integrons that confer resistance to multiple antibiotics; various sizes between 0.6 and 3.1 kb were found. None of the isolates contained Class II integrons. Our results indicate that farm-raised catfish may be a source of pathogenic A. veronii and that the potential health risks posed by virulent strains of A. veronii should not be underestimated.     

Antimicrobial activity of enterococci strains isolated from white cheese

Twelve strains of enterococci isolated from white cheese samples obtained from different regions of Turkey were screened for their antimicrobial activity against some Gram-positive and Gram-negative food-borne pathogens and contaminating bacteria, also against themselves and some other lactic acid bacteria (LAB), including some species of lactococci, lactobacilli and Leuconostoc . Antimicrobial activity was confirmed for all 12 strains, including six Enterococcus faecium , two Enterococcus faecalis and four Enterococcus durans . Antimicrobial agents produced by these enterococci exhibited a spectrum of activity, which is mainly directed against food-borne pathogens, specially Listeria monocytogenes and Bacillus cereus and some other Bacillus spp. and also some enterococci. Tested LAB was insensitive to inhibitory agents produced by them.     

Microbiological study of semi-hard goat's milk cheese (Majorero)

The development of microbial flora in industrially produced semi-hard cheese made from pasteurized goat's milk was studied during manufacture and over a 90-day ripening period.
Estimates of total count, streptococci, lactobacilli, leuconostocs, coliforms, micrococci and staphylococci, Staphylococcus aureus and yeasts and moulds were carried out at various stages of the ripening process; streptococci and lactobacilli were identified by species.
Initially, the total count increased rapidly, primarily as a result of the growth of mesophilic lactic streptococci mainly Streptococcus lactis and Strep, cremoris. Subsequently, both these counts stabilized or decreased. Lactobacilli increased, and by the end of the ripening period were the predominant microorganisms. Most common were Lactobacillus casei var. casei. , especially at the end of storage; L. casei var. rhamnosus , L. casei var. plantarum and L. cellobiosus were also isolated. Leuconostocs were not found in any of the cheeses, and hence no eye formation took place. Coliforms, enterococci, yeasts and moulds remained below 10²–10³ c.f.u. g⁻¹. Maximum levels of micrococci and staphylococci were found after 15–30 days of ripening and decreased gradually towards the end of the ripening period. Neither the milk curd, nor cheese contained Staph. aureus.     

Putative virulence properties of Aeromonas strains isolated from food, environmental and clinical sources in Italy: a comparative study

The distribution of virulence properties in 142 strains of Aeromonas isolated from diarrhoeic patients, food and surface water in Italy and identified by biochemical and molecular methods was investigated. The virulence properties studied were the presence of genes for the aerolysin (aerA), heat-stable cytotonic enterotoxin (ast), heat-labile cytotonic enterotoxin (alt), cytotoxic enterotoxin (act); and cytotoxicity for Vero cells and adhesion on Hep-2 cells. A. hydrophila and A. caviae were the species most commonly isolated from clinical and environmental samples (9/30; 30.0% and 5/27; 18.5%, respectively) while mesophilic A. salmonicida was most common in food samples (19/80; 23.7%). Out of 142 strains, 86 (60.6%) were positive for at least one of the virulence properties. All the toxin genes were present in 4/18 (22.3%) of clinical strains. Most of the food isolates (54/55; 98.2%) were cytotoxic and most of the environmental strains (12/13; 92.3%) were adhesive. The aerA gene was present in most toxigenic strains (72/86; 83.7%), irrespective of their origin. The growth temperature affected the expression of cytotoxicity and adhesivity. Aeromonas strains from food and surface water frequently had toxin gene patterns similar to those of clinical strains and expressed virulence properties at human body temperature. These findings indicate that aeromonads have the potential to cause human illness and confirm the role of food and water as vehicles for Aeromonas diseases.     

Characterization for enterotoxin production,virulence factors,and antibiotic susceptibility of Staphylococcus aureus isolates from various foods in Portugal

Staphylococcus aureus represents a public health challenge worldwide. The aim of this study was the characterization of different food isolates of S. aureus on the basis of their production of enterotoxins, hemolysins and resistance to antibiotics. A total of 148 coagulase-positive staphylococcal strains isolated from different food origins were identified to the species level. By multiplex PCR, 69% of the isolates were shown to be enterotoxigenic (SEs); the most common were sea seg, sea seg sei and seg sei. According to CLSI [CLSI, Clinical and Laboratory Standards Institute, 2007. Performance Standards for Antimicrobial Susceptibility Testing; Fifteenth Informational Supplement. CLSI document M100-S15. Clinical and Laboratory Standards Institute, Wayne, PA], 38% of the isolates were resistant to oxacillin (≥6 μg/mL; MRSA positives) but only 0.68% showed the presence of mecA gene. 70 and 73% of the S. aureus strains were resistant to β-lactams, ampicillin and penicillin, respectively. The virulence pattern was demonstrated to be origin and strain dependent. These findings emphasise the need to prevent the presence of S. aureus strains and SEs production in foods.     

冷藏草鱼源气单胞菌的群体感应现象及其对生物膜形成的影响

以冷藏草鱼的优势腐败菌气单胞菌（Aeromonas spp.）为研究对象,利用报告菌株Chromobacterium violaceum CV026检测细菌的群体感应活性,通过染色法定量测定细菌的生物膜形成,并探究温度以及群体感应信号分子对气单胞菌生物膜形成的影响。结果显示,冷藏草鱼的气单胞菌属包含多个不同种,分离获得的20 株气单胞菌中90%能产生N-酰基高丝氨酸内酯。进一步分析生物膜形成量较强的2 株菌A. salmonia W41和A. salmonia W69,结果显示该2 株菌在30 ℃和8 ℃的生物膜形成量高于37 ℃。菌株W41和W69主要分泌N-丁酰基高丝氨酸内酯（N-butanoyl-L-homoserine lactone,C4-HSL）和N-己酰基高丝氨酸内酯（N-hexanoyl-L-homoserine lactone,C6-HSL）信号分子,50 μmol/L的C6-HSL显著促进W41生物膜的形成;而低浓度的C4-HSL和C6-HSL促进W69生物膜的形成,高浓度的C4-HSL和C6-HSL抑制W69生物膜的形成。可以看出,大多数气单胞菌属具有群体感应现象,气单胞菌属生物膜的形成受个体差异和温度的影响,同时群体感应信号分子的浓度和类型也是影响气单胞菌属生物膜形成的主要因素。     

Detection of potentially pathogenic Aeromonas isolates from ready‐to‐eat seafood products by PCR analysis

This study provides data on the prevalence of potentially pathogenic Aeromonas spp. in ready‐to‐eat (RTE) seafood products by evaluating the occurrence of Aeromonas spp. and the presence of virulence‐associated genes. Aeromonas spp. was detected in 57/81 (70.3%) RTE seafood samples. Specifically, Aeromonas spp. was highlighted in 19/21 (90.5%) sushi, in 18/21 (85.7%) sea salad, 11/12 (91.7%) surimi and 9/12 (75%) peeled shrimp samples. Aeromonas spp. was not observed in marinated anchovies and octopus salad samples. Then, PCRs aimed at the hlyA, aerA, alt and ast genes, encoding, respectively, haemolysin A, aerolysin, aeromonas labile temperature cytotonic enterotoxin and aeromonas stable temperature cytotonic enterotoxin, demonstrated a widespread distribution of these genes among Aeromonas isolates. The results underline the need to implement an adequate control plan performing an intensive and continuous monitoring to guarantee the human health.     

Physicochemical,textural and sensory properties of white soft cheese made from buffalo and cow milk mixtures

The physicochemical, textural and sensory properties of white soft cheeses made from three different buffalo and cow milk mixtures (100:0, 70:30 and 30:70) during 3‐month storage were studied. The increase in buffalo milk concentration resulted in increasing total cheese yield, dry matter (DM) and fat retention and fat in DM content. However, it caused reductions in moisture content, salt intake, hardness, chewiness, elasticity, sensory hardness and sensory cohesiveness of the samples. The percentage of water‐soluble nitrogen to total nitrogen increased during storage resulting in decreased fracturability, hardness (textural and sensory), cohesiveness (textural and sensory), springiness, chewiness and elasticity. The panellists evaluated the white soft cheese made with buffalo milk as the most acceptable.     

不同场景下冷却猪肉中气单胞菌到小青菜的交叉污染

为了定量分析厨房内冷却猪肉中气单胞菌（Aeromonas spp.）在案板、刀具、手以及小青菜之间的交叉污染水平,采用一定方式将气单胞菌接种于精腿肉中,模拟消费者在厨房中食物准备过程,对猪肉进行分割,分别测定气单胞菌到案板、刀具、手的转移率;同时,测定6 种设定场景下接种于3 种介质表面的气单胞菌到小青菜的转移率,并通过对数转换后进行频数分布拟合。此外,以场景1即切过猪肉后各介质不做任何处理的情况为例对交叉污染进行仿真模拟。结果表明：各组转移率在一定范围内变化且差异显著,经对数转换后的转移率符合或接近正态分布。交叉污染的仿真模拟证实,若食用产生交叉污染的猪肉和蔬菜将存在一定的风险,再结合烹饪阶段的评估以及剂量-反应关系研究,可为构建完整气单胞菌风险评估体系提供理论参考。     

猪肉解冻过程中损伤型气单胞菌的检测及失活模型构建

研究猪肉在解冻过程中其表面的损伤型气单胞菌检测和生长模型的构建。以含质量分数0.6%酵母浸膏的胰酪胨大豆琼脂(TSAYE)为选择培养基,气单胞菌的损伤培养基中分别添加质量分数1.0%、1.5%、2.0%、2.5%、3.0%的NaCl,经差异显著性分析得出TSAYE+1.5% NaCl作为合适损伤培养基并用于后续研究。损伤型气单胞菌的数量采用普通培养基的菌落数与损伤培养基的菌落数之差来计数。猪肉在25℃解冻过程中,损伤型气单胞菌的修复时间大约为75min,其失活符合Polynomial模型(R2＝0.9937)及Boltzmann模型(R2＝0.9884),通过模型的检验选择Polynomial模型拟合损伤型气单胞菌的失活过程。     

Effect of starter cultures on properties of soft white cheese made from camel (Camelus dromedarius) milk

This experiment was conducted to investigate the effect of starter cultures on the physicochemical properties, texture, and consumer preferences of soft white cheese (SWC) made from camel (Camelus dromedarius) milk. The experiment was laid out in a completely randomized design with 5 treatments [starter cultures; i.e., 1 thermophilic (STI-12), 2 blended (RST-743 and XPL-2), and 2 mesophilic (R-707 and CHN-22) cultures]. Starter cultures STI-12 and RST-743 were inoculated at 37°C, whereas XPL-2, R-707, and CHN-22 were inoculated at 30°C. Camel milk inoculated using STI-12 and RST-743 cultures resulted in faster acidification than XPL-2, R-707, and CHN-22 cultures. Camel milk SWC made using STI-12 and CHN-22 cultures gave lower pH (4.54) and titratable acidity (0.59), respectively, whereas R-707 culture resulted in high cheese yield (13.44 g/100 g). In addition, high fat (20.91 g/100 g), protein (17.49 g/100 g), total solids (43.44 g/100 g), and ash (2.40 g/100 g) contents were recorded for SWC made from camel milk made using RST-743 culture. Instrumental analysis of cheese texture revealed differences in resistance to deformation in which camel milk SWC made using RST-743 culture gave higher firmness (3.20 N) and brittleness (3.12 N). However, no significant difference was observed among camel milk SWC adhesiveness made using different starter cultures. Consumer preference for appearance, aroma, taste, and overall acceptances of SWC were affected by inoculation of starter cultures. Considering curd firmness, cheese yield, compositional quality, and textures using STI-12, RST-743, and R-707, these cultures were found to be better for the manufacture of camel milk SWC.     

Production and properties of a semi-hard cheese made from soya milk

Summary A semi-hard soya cheese, with mean moisture content 61.5%, crude protein 21.8% and fat 2.6%, was produced from reconstituted soya-milk powder using a starter culture of Streptococcus thermophilus and Lactobacillus fermentum . The physical properties of the cheese, as determined with a Texture Profile Analyser , were similar to a cheese made to the same compositional standards from bovine milk. A taste panel of Far Eastern subjects did not find the flavour of the fresh soya cheese acceptable but, when cubes of the cheese (1 cm³) were deep-fried in corn oil, the hedonic rating improved significantly. It is suggested that the cheese could be used as a protein-rich component of a meal, e.g. to replace meat in a stew, or as a 'snack food'.     

冷却猪肉中气单胞菌暴露评估的不确定性和变异性

探讨冷却猪肉中气单胞菌暴露评估是否区分不确定性和变异性对结果的影响,并基于前期评估结果,对冷却猪肉中气单胞菌从销售到食用前阶段的增殖情况通过点估计和概率分布估计两种方法来实现。结果表明：点估计可能低估食用风险,概率分布考虑了输入量的各种可能性,推测结果更符合实际情况;同时,以符合正态分布的家庭贮藏温度为例修正参数设置,考虑全为不确定性,部分不确定性、部分变异性,全为变异性3 种可能情况,得到区分不确定性和变异性后的风险概率由不区分前的22.1%降为20.2%,全为变异性的概率仅为11.0%,从方法论角度探讨不同方法获得的风险差异,为后续定量风险评估提高精确性和可靠性提供理论参考。     

Biodiversity and characterization of Staphylococcus species isolated from a small manufacturing dairy plant in Portugal

The level and the diversity of the staphylococcal community occurring in the environment and dairy products of a small manufacturing dairy plant were investigated. Species identification was performed using different molecular methods, viz. Multiplex-PCR, amplified ribosomal DNA restriction analysis (ARDRA), and sodA gene sequencing. The main species encountered corresponded to Staphylococcus equorum (41 isolates, 39.0%), S. saprophyticus (28 isolates, 26.7%) and S. epidermidis (15 isolates, 14.3%). Additionally, low incidence of enterotoxin genes was obtained, with only 9 strains (8.6%) being positive for one or more toxin genes. With regard to antimicrobial resistance, 57.1% of the isolates showed at least resistance against one antibiotic, and 28.6% were multi-resistant, which might accomplish resistance for up to 6 antibiotics simultaneously. These results provided evidence that the presence of Staphylococcus species in dairy environment are mostly represented by S. equorum and S. saprophyticus, and illustrate that carrying antimicrobial resistance genes has become reasonably widespread in cheese and dairy environment.     

Some physicochemical, microbiological, and sensory properties of tulum cheese produced from ewe's milk via a modified method

The purpose of this research was to investigate an alternative way to manufacture Erzincan tulum cheese in order to shorten production time and improve food safety. By adding 0.5% starter culture ( Streptococcus salivarius ssp. thermophilus and Lactobacillus delbrueckii ssp. bulgaricus cultures at a 1 : 1 ratio) to pasteurized ewe's milk (65°C for 30 min), the required time for manufacturing Erzincan tulum cheese was shortened from the traditional 10–12 days to 2 days. The cheeses manufactured with the modified method were ripened in three different packaging materials: goatskin, plastic, and ceramic. Physicochemical, microbiological, and sensory properties of the Erzincan tulum cheese were obtained during the ripening period at intervals of 2, 30, 60, and 90 days, and compared with those properties of samples manufactured by the traditional method. Significant microbiological and physicochemical differences were found between the modified samples and the traditional samples ( P <  0.01). However, the modified samples and the traditional samples were statistically similar in sensory properties to the exception of the modified sample packaged in plastic.     

Prevalence, characterization, and antimicrobial resistance of Aeromonas strains from various retail food products in Mumbai, India

A total of 154 food samples (chicken, fish, and ready-to-eat sprouts) from various retail outlets in Mumbai, India, were analyzed for the presence of Aeromonas spp. over a period of 2 y (January 2006 to March 2008). Twenty-two Aeromonas isolates belonging to 7 different species were isolated from 18 (11.7%) food samples. The highest percentages of isolation were from chicken (28.6%) followed by fish (20%) and sprout (2.5%) samples. Aeromonas caviae, A. veronii bv. sobria, and A. salmonicida were the most frequently isolated species from sprouts, chicken, and fish samples, respectively. The genes encoding for putative virulence factors, cytotoxic enterotoxin (act), hemolysin (hly), aerolysin (aer), elastase (ahyB), and lipase (lip) were detected using polymerase chain reaction method in 59.1%, 40.9%, 22.7%, 54.5%, and 31.8% of the strains, respectively. The isolated Aeromonas strains were found to be positive for virulence factors, that is, amylase, DNase, gelatinase, protease, and lipase production. More than 60% isolates were also positive for β-hemolytic activity. All these food isolates were found to be resistant to ampicillin and bacitracin, and sensitive to gentamicin, 3rd-generation cephalosporins (ceftazidime, cephotaxime, ceftriaxone), and chloramphenicol. Seventeen (77.2%) isolates harbored single and/or multiple plasmids (approximately 5 to >16 kb). The XbaI digestion patterns of chromosomal DNA of these isolates, using pulsed field gel electrophoresis, showed high genetic diversity among these isolates. Our results demonstrate the presence of various Aeromonas spp. with virulence potential and antimicrobial resistance in different food products marketed in Mumbai, India. The potential health risks posed by consumption of these raw or undercooked food products should not be underestimated.     

Biochemical,microbial and sensory evaluation of white soft cheese made from cow and lupin milk

The aim of the present study was to evaluate some physicochemical, microbiological and sensory properties of fresh and matured (75 days) soft cheeses made with mixtures of cow milk and 0, 25, 50 and 75 mL/100 mL of lupin milk. A remarkable increase in cheese yield was observed with increasing the level of lupin milk to the mixture. Compared to cow milk cheese, the protein content was significantly (P ≤ 0.05) increased while ash was decreased with the increase in the level of lupin milk for both fresh and matured cheese. However, fat content, total solids and acidity were increased only for fresh cheese and decreased for mature one compared to that of cow milk. The pH showed significant (P ≤ 0.05) reduction when the levels of lupin milk increased for fresh cheese while for matured cheese it slightly decreased. The total bacterial count is within the range that naturally exists in milk containing foods. The others microorganisms such as fungi, mold, Escherichia coli, and Salmonella were not existed in both types of cheese. Regardless of cheese color, incorporation of lupin milk at low concentration (25 mL/100 mL) significantly (P ≤ 0.05) enhanced the taste, texture, flavor, and overall acceptability of both fresh and mature cheese.     

Genotypic and phenotypic virulence characteristics and antimicrobial resistance of Yersinia spp. isolated from meat and milk products

A total of 300 food samples including 180 milk and 120 meat products have been examined for the presence of Yersinia spp. using the ISO 10273 and the cold enrichment method. The overall prevalence of Yersinia spp. was 84 (28%). Yersinia enterocolitica was isolated from 18 (6%) of the 300 samples. The other Yersinia species were detected in the samples Yersinia rohdei 15 (5%), Yersinia intermedia 14 (4.7%), Yersinia pseudotuberculosis 12 (4%), Yersinia ruckeri 12 (4%), Yersinia mollaretii 5 (1.7%), Yersinia bercovieri 4 (1.3%), and atypical Yersinia spp. 4 (1.3%). The conventionally identified Y. enterocolitica strains were also confirmed by the 16S rRNA gene sequencing. All Y. enterocolitica strains biotyped as 1A had negative results in the phenotypic virulence tests. The 84 Yersinia strains were also examined genotypically for the presence of virulence genes. None of the Y. enterocolitica and other Yersinia strains contained the ail, ystA, yadA, and virF except only 1 Y. intermedia and 2 Y. enterocolitica strains that were found to be positive for ystB. Antimicrobial resistance of 84 Yersinia to 16 antimicrobial agents was determined by the disk diffusion method. All strains were sensitive to tobramycine and imipenem while resistant to clindamycin. Although 84.5% of the strains were resistant to at least 3 or more antimicrobial agents, 64.3% of them were resistant to 4 or more antimicrobial agents.     

Prevalence and characterization of Enterococcus spp. isolated from Brazilian foods

Enterococci can be used in the food industry as starter or probiotic cultures. However, enterococci are also implicated in severe multi-resistant nosocomial infections. In this study, the prevalence of enterococci in selected Brazilian foodstuffs (raw and pasteurized milk, meat products, cheeses and vegetables) was evaluated. Phenotypic and PCR protocols were used for species identification. Tests for production of gelatinase, haemolysin, bacteriocin and bile salt hydrolysis were done with all enterococci isolates, whereas molecular determination of virulence markers (genes esp, gel, ace, as, efaA, hyl and cylA) and antibiotic resistance was checked only for Enterococcus faecium and Enterococcus faecalis isolates. The antibiotic-resistant isolates were assayed for biofilm formation and adhesion to mammalian cells. From the 120 food samples analyzed, 52.5% were positive for enterococci, meat and cheese being the most contaminated. E. faecium was the predominant species, followed by E. faecalis, E. casseliflavus and Enterococcus gallinarum. Phenotypic tests indicated that 67.7% of isolates hydrolyzed bile salts, 15.2% produced bacteriocin, 12.0% were beta-hemolytic and 18.2% produced gelatinase. Antibiotic resistance (gentamicin, tetracycline and erythromycin) and genes encoding for virulence traits were more frequent in E. faecalis than in E. faecium. Three E. faecium isolates were resistant to vancomycin. Among antibiotic-resistant isolates, 72.4% of E. faecalis were able to form biofilm and 13.8% to adhere to Caco-2 cells. Antibiotic-resistant E. faecalis and E. faecium isolates were grouped by RAPD-PCR and a scattered distribution was noted, indicating that resistance was not related to a particular clone. The spread of virulence/resistance traits in isolates of the two species and different RAPD-types suggest the pathogenic potential of both species. By contrast, the recovery of bacteriocinogenic E. faecium isolates with no virulence traits suggests their potential for biotechnological applications. In conclusion, our results showed that enterococci from Brazilian foods present important dualist aspects for food safety.     

齐口裂腹鱼肠道气单胞菌的分离鉴定

本研究从健康齐口裂腹鱼肠道内分离到3株气单胞菌株,采用单重PCR及多重PCR技术进行鉴定。结果表明,16S rRNA扩增长度为350bp,与Gen Bank数据库中的基因序列进行比对发现其与亚洲地区已报道的同类基因片段相似度均达到了90%以上。气单胞菌气溶素基因(aer)和嗜水气单胞菌溶血素基因(AHH)扩增长度分别为300bp和130bp,而温和气单胞菌溶血素基因(ASA)未扩增到条带。通过基因序列比对发现,aer和ASA与已报道的嗜水气单胞菌的对应的基因片段相似度均达到90%以上。结论,从健康齐口裂腹鱼肠道内所分离的3株气单胞菌菌株,均为致病性嗜水性气单胞菌。