Molecular characterisation,genotyping and survival of Aeromonas hydrophila isolated from milk,dairy products and humans in Egypt

Two Aeromonas species, Aeromonas hydrophila and Aeromonas sobria, were isolated from raw milk (8% and 5.3% of samples tested, respectively), yoghurt (12% and 8% of samples tested, respectively) and cheese (4% and 2% of samples tested, respectively). Only A. hydrophila was isolated from human stool samples (18.8% of samples tested). Aerolysin and haemolysin associated genes were characterised in 12 and 3 isolates, respectively, while both genes were identified simultaneously in 9 isolates. Genotyping of the isolates by random amplified polymorphic DNA (RAPD) PCR revealed a high discriminatory index (D = 0.966). The storage of yoghurt samples inoculated with A. hydrophila showed the ability of the bacteria to survive for 14 days, resembling the shelf-life of yoghurt at 4 °C and 12 °C. To the best of our knowledge, this is the first study to assess the survival of A. hydrophila in yoghurt at refrigeration temperature.     

Growth of and toxin production by Aeromonas hydrophila and Aeromonas sobria at low temperatures

The effects of different temperatures on the growth and toxin production of Aeromonas hydrophila and Aeromonas sobria were studied. The results showed that these Aeromonas species are not only able to grow at low temperatures (e.g. at 4 and 10 degrees C) but may also produce cytotoxin, hemolysin and enterotoxin under suitable growth conditions.     

Aflatoxins in dairy cow feed,raw milk and milk products from Turkey

This study aims to detect aflatoxins (AFs) in dairy cow feed, milk and milk products using a high-performance liquid chromatography coupled with fluorescence detection (HPLC-FLD) method. All the validation parameters met the method performance criteria of the European Union. The samples comprised 76 dairy cow feeds and 205 milk and milk products (including yoghurt and yoghurt-based beverage, ayran). AFs were present in 26.3% of the feed samples. Two feed samples exceeded the maximum limit (ML) of 5 µg kg^?1 for AFB₁ as established by the EU. Nineteen milk samples (21.1%) contained aflatoxin M₁ (AFM₁) of which three exceeded the EU ML of 0.05 µg l^?1. In addition, only two yoghurt samples and one ayran sample contained AFM₁, but the levels were lower than the EU ML.     

Experimental evidence for toxin production by Aeromonas hydrophila and Aeromonas sobria in a meat extract at low temperatures

The ability of enterotoxigenic strains of Aeromonas hydrophila and Aeromonas sobria to produce exotoxins (enterotoxin and haemolysin) in a meat extract at low temperatures (5 and 12 degrees C) was investigated. All three strains incubated at 12 degrees C were enterotoxigenic and haemolytic in the meat extract after 5 days. At 5 degrees C, five of the six strains tested were able to produce these exotoxins after 8 days incubation while one strain was neither enterotoxigenic nor haemolytic after 5, 8 and 11 days. The possible involvement of performed toxin(s) in Aeromonas gastroenteritis is also discussed.     

Development of a multiplex and semi-nested PCR assay for detection of Yersinia enterocolitica and Aeromonas hydrophila in raw milk

Single-locus (sl), multiplex (m), and semi-nested (sn) polymerase chain reaction (PCR) procedures were developed for the detection of Yersinia enterocolitica and Aeromonas hydrophila in raw milk samples. Two oligonucleotide primers for each pathogen were used in PCR, to detect the yst gene of Y. enterocolitica and aer gene of A. hydrophila. The amplified fragment size was 145 base-pair (bp) for yst gene, and 209 bp for the aer gene. The performance of the systems were evaluated with seeded milk samples, and naturally contaminated raw milk samples. PCR results were compared with conventional cultural procedures. The limits of detection of slPCR and mPCR assays were approximately 10² cfu ml⁻¹ (0·5 cfu/PCR reaction mixture) for both pathogens in seeded raw milk. When studied with naturally contaminated raw milk samples, detection rates obtained by PCR and cultural methods were 53% and 36% for Y. enterocolitica and were 23% and 14% for A. hydrophila, respectively. These results indicate that the direct PCR analysis of raw milk can be used as a rapid and specific diagnostic method for both pathogens.     

Hemolytic and proteolytic activities of Aeromonas hydrophila and Aeromonas veronii biovar sobria in broth and salmon extract at different temperatures

Expression of hemolytic and proteolytic activities throughout the growth cycle was investigated with two enterotoxic aeromonad strains assigned to the species Aeromonas hydrophila and Aeromonas veronii biovar sobria. Although growth kinetic data were dependent on strain, temperature, and substrate, maximum populations attained were higher than 9 log CFU/ml in aerated tryptone soya broth plus yeast extract (TSBYE) and salmon extract within the range 4 to 28 degrees C. For both strains in TSBYE, variable amounts of hemolytic activity were first detected at any temperature when aeromonad counts were over 9 log CFU/ml. Afterwards, this activity increased up to similar levels (109 to 112 hemolytic units per ml) without a significant increase in populations. Salmon extract supported hemolysin synthesis at 28 but not 4 degrees C. Proteolytic activity of the A. hydrophila strain was only expressed in salmon extract at 28 degrees C, whereas A. veronii biovar sobria did at 28 degrees C in both substrates and at 10 degrees C in TSBYE.     

Growth of Aeromonas hydrophila in modified-atmosphere-packed cooked meat products

Growth of Aeromonas hydrophila was investigated in modified-atmosphere-packed cooked meat products by developing predictive models. Modified brain–heart infusion (BHI) was shown to be suitable as a simulation medium for cooked meat products. Predictive models were developed for the growth parameters (maximum specific growth rate and lag phase) of A. hydrophila in modified BHI as a function of temperature, water activity and concentration of dissolved carbon dioxide. The growth of A. hydrophila was compared with the growth of the Specific Spoilage Organism (SSO) for cooked meat products Lactobacillus sake, to determine possible risk areas for A. hydrophila in modified atmosphere-packed cooked meat products. Aeromonas hydrophila was shown to multiply very rapidly at refrigerated temperatures. The developed models clearly demonstrated however that proliferation of A. hydrophila could be prevented by the use of carbon dioxide in the package atmosphere in combination with a decreased water activity (<0·985). Gas-packed cured cooked meat products will not sustain the growth of A. hydrophila when kept at refrigerated temperatures (<7°C).     

Identification of proteolytic pseudomonads isolated from raw milk

Proteolytic pseudomonads isolated from raw milk were classified by numerical taxonomy. Unweighted pair-group average-linkage cluster analysis was used to cluster 49 bacterial strains, of which 26 were Pseudomonas species, as described in the Bergey's Manual of Determinative Bacteriology, based on 52 characters. The milk isolates resided in two clusters; one containing Pseudomonas fluorescens and the other Pseudomonas fragi. The isolates identified with Pseudomonas fluorescens hydrolyzed milk proteins and milk fat and produced phospholipase. The Pseudomonas fragi-like isolates hydrolyzed milk proteins and milk fat but did not produce phospholipase and fluorescent pigment. The hydrolytic characteristics of milk isolates showed that the nature of the substrate and conditions under which the test was conducted were critical.     

Enumeration and identification of yeasts isolated from Zimbabwean traditional fermented milk

The yeasts in 30 samples of the Zimbabwean traditional fermented milk, amasi, taken from farms, households and milk collection centres were enumerated and identified. The yeast counts ranged from <2 to 8.08 log cfu g⁻¹. Yeast isolates were identified using the API ID 32°C test kits and the simplified identification method (SIM) as well as with reference to the standard taxonomic keys. From the 30 samples, a total of 20 different yeast species were identified. Saccharomyces (S) cerevisiae (22 isolates), Candida (C) lusitaniae (11), C. colliculosa (7) and S. dairenensis (7) were the predominant species identified. Dekera (Dek.) bruxillensis, C. lipolytica and C. tropicalis were identified less often. Seven of the S. cerevisiae isolates were able to assimilatedl-lactate. The strain of C. kefyr isolated could assimilate lactose anddl-lactate, but not citrate. The analysed amasi samples contained a wide variety of yeasts, but only a few species predominated and these could possibly contribute to the characteristics of the fermented milk in the 48 h fermentation.     

Prevalence of Arcobacter spp. in raw milk and retail raw meats in Northern Ireland

A 1-year study was undertaken to determine the prevalence of Arcobacter spp. in raw milk and retail raw meats on sale in Northern Ireland. Retail raw poultry samples (n = 94), pork samples (n = 101), and beef samples (n = 108) were obtained from supermarkets in Northern Ireland, and raw milk samples (n = 101) were kindly provided by the Milk Research Laboratory, Department of Agriculture and Rural Development, Belfast, Northern Ireland. Presumptive arcobacters were identified by previously described genus-specific and species-specific PCR assays. Arcobacter spp. were found to be common contaminants of retail raw meats and raw milk in Northern Ireland. Poultry meat (62%) had the highest prevalence, but frequent isolations were made from pork (35%), beef (34%), and raw milk (46%). Arcobacter butzleri was the predominant species isolated from retail raw meats and was the only species isolated from raw milk samples. Arcobacter cryaerophilus was detected less frequently, and Arcobacter skirrowii was detected only as a cocontaminant. To our knowledge, this is the first report of Arcobacter spp. prevalence in a diverse range of products of animal origin in Northern Ireland.     

Polybrominated biphenyls in raw milk and processed dairy products.

Milk from four dairy herds identified by the Michigan Department of Agriculture as containing less than .3 ppm (fat basis) physiologically incorporated polybrominated biphenyls was processed individually into cream, skim milk, butter, and stirred curd cheese. Pasteurized and freeze-dried whole milk, skim milk, and cream, spray-dried whole milk and skim milk, and condensed whole milk were made also. Polybrominated biphenyls were concentrated in the high-fat products. Pasteurized skim milk, buttermilk, and whey had slightly more polybrominated biphenyls than pasteurized whole milk on a fat basis. Spray-drying reduced the polybrominated biphenyls in whole milk and skim milk while pasteurization, freeze-drying, aging of cheese, and condensation were not effective.     

Occurrence, virulence genes and antibiotic resistance of Escherichia coli O157 isolated from raw bovine, caprine and ovine milk in Greece

The examination of 2005 raw bovine (n = 950), caprine (n = 460) and ovine (n = 595) bulk milk samples collected throughout several regions in Greece for the presence of Escherichia coli serogroup O157 resulted in the isolation of 29 strains (1.4%) of which 21 were isolated from bovine (2.2%), 3 from caprine (0.7%) and 5 from ovine (0.8%) milk. Out of the 29 E. coli O157 isolates, only 12 (41.4%) could be classified as Shiga-toxigenic based on immunoassay and PCR results. All 12 Shiga-toxigenic E. coli serogroup O157 isolates belonged to the E. coli O157:H7 serotype. All except one of the 12 Shiga-toxin positive isolates were stx₂-positive, five of which were also stx₁-positive. The remaining isolate was positive only for the stx₁ gene. All stx-positive isolates (whether positive for stx₁, stx₂ or stx₁ and stx₂) were also PCR-positive for the eae and ehxA genes. The remaining 17 E. coli O157 isolates (58.6%) were negative for the presence of the H7 flagellar gene by PCR, tested negative for Shiga-toxin production both by immunoassay and PCR, and among these, only four and three strains were PCR-positive for the eae and ehxA genes, respectively. All 29 E. coli O157 isolates displayed resistance to a wide range of antimicrobials, with the stx-positive isolates being, on average, resistant to a higher number of antibiotics than those which were stx-negative.     

Antibiotic resistance of Aeromonas hydrophila isolated from marketed fish and prawn of South India

From February to November 1999, 198 samples of chicken meat for sale in retail outlets and supermarkets in nine provinces of Castilla and León (Spain) were analysed for the prevalence of Salmonella and Campylobacter. Salmonella was isolated from 71 (35.83%) of the samples analysed. The predominant serovars were S. enteritidis (47.88%), S. hadar (25.35%) and serotype 4,12:b:-(II) (19.71%). Other serovars such as S. mbandaka, S. derby, S. virchow and S. paratyphi B were isolated in much lower levels. Thermophilic campylobacters were isolated in 49.50% of the samples studied.     

Genetic diversity of Lactobacillus delbrueckii isolated from raw milk in Hokkaido,Japan

Lactic acid bacteria (LAB) play important roles in acid production and flavor formation in fermented dairy products. Lactic acid bacteria strains with distinct characteristics confer unique features to products. Diverse LAB have been identified in raw milk and traditional fermented milk prepared from raw milk. However, little is known about LAB in raw milk in Japan. To preserve diverse LAB as potential starters or probiotics for future use, we have isolated and identified various kinds of LAB from raw milk produced in Japan. In this study, we focused on Lactobacillus delbrueckii, one of the most important species in the dairy industry. We identified L. delbrueckii subspecies isolated from raw milk in Hokkaido, Japan, by analyzing intraspecific diversity using 4 distinct methods, hsp60 cluster analysis, multilocus sequence analysis, core-genome analysis, and whole-genome analysis based on average nucleotide identity. The subspecies distribution and a new dominant subset of L. delbrueckii from raw milk in Japan were revealed. The discovery of new strains with different genotypes is important for understanding the geographic distribution and characteristics of the bacteria and further their use as a microbial resource with the potential to express unconventional flavors and functionalities. The strains identified in this study may have practical applications in the development of fermented dairy products.     

The analysis of milk components and pathogenic bacteria isolated from bovine raw milk in Korea

Bovine mastitis can be diagnosed by abnormalities in milk components and somatic cell count (SCC), as well as by clinical signs. We examined raw milk in Korea by analyzing SCC, milk urea nitrogen (MUN), and the percentages of milk components (milk fat, protein, and lactose). The associations between SCC or MUN and other milk components were investigated, as well as the relationships between the bacterial species isolated from milk. Somatic cell counts, MUN, and the percentages of milk fat, protein, and lactose were analyzed in 30,019 raw milk samples collected from 2003 to 2006. The regression coefficients of natural logarithmic-transformed SCC (SCCt) on milk fat (−0.0149), lactose (−0.8910), and MUN (−0.0096), and those of MUN on milk fat (−0.3125), protein (−0.8012), and SCCt (−0.0671) were negative, whereas the regression coefficient of SCCt on protein was positive (0.3023). When the data were categorized by the presence or absence of bacterial infection in raw milk, SCCt was negatively associated with milk fat (−0.0172), protein (−0.2693), and lactose (−0.4108). The SCCt values were significantly affected by bacterial species. In particular, 104 milk samples infected with Staphylococcus aureus had the highest SCCt (1.67) compared with milk containing other mastitis-causing bacteria: coagulase-negative staphylococci (n = 755, 1.50), coagulase-positive staphylococci (except Staphylococcus aureus; n = 77, 1.59), Streptococcus spp. (Streptococcus dysgalactiae, n = 37; Streptococcus uberis, n = 12, 0.83), Enterococcus spp. (n = 46, 1.04), Escherichia coli (n = 705, 1.56), Pseudomonas spp. (n = 456, 1.59), and yeast (n = 189, 1.52). These results show that high SCC and MUN negatively affect milk components and that a statistical approach associating SCC, MUN, and milk components by bacterial infection can explain the patterns among them. Bacterial species present in raw milk are an important influence on SCC in Korea.     

Genetic diversity of Gram-negative, proteolytic, psychrotrophic bacteria isolated from refrigerated raw milk

The random amplified polymorphic DNA (RAPD) fingerprinting technique was used to assess the genetic diversity of 70 isolates of Gram-negative proteolytic psychrotrophic bacteria that were isolated from refrigerated raw milk. Three oligonucleotides, which generated 87 fragments of polymorphic DNA, were used in the amplification reactions. The genetic distance values calculated using Jaccard's coefficient showed there was high genetic variability among the isolates. Cluster analysis procedures suggested that the genetic variability among isolates belonging to the same species was as high as the variability among different species. Clustering by the UPGMA hierarchical method and data graph dispersion indicated a tendency of the isolates to group according to whether they did or did not ferment glucose.     

Antihypertensive activity of milk fermented by Enterococcus faecalis strains isolated from raw milk

A total of 231 microorganisms were isolated from raw cow milk samples and the angiotensin-converting enzyme-inhibitory (ACEI) activity of the resultant fermented milk produced with the isolated microorganisms was assayed. Forty-six of these microorganisms were selected on the basis of high ACEI activity. Four Enterococcus faecalis strains stood out as producers of fermented milk with potent ACEI activity (IC₅₀ (the protein concentration that inhibits 50% of ACE activity): 34–59 μg mL⁻¹). Single doses (5 mL kg⁻¹) of the whey fraction obtained from these fermented milk samples were administered to spontaneously hypertensive rats (SHR) and to normotensive Wistar-Kyoto (WKY) rats in order to investigate their possible antihypertensive activity. Highly significant decreases in the systolic blood pressure (SBP) and in the diastolic blood pressure (DBP) were observed when the fermented milk was administered to SHR. Nevertheless, the fermented milk did not modify the SBP and the DBP of the WKY rats. Raw cow milk is an excellent source of wild lactic acid bacteria able to produce fermented milk with antihypertensive activity and antihypertensive activity of milk fermented by Enterococcus faecalis strains was associated with peptides different from Ile-Pro-Pro and Val-Pro-Pro.     

原料乳与乳制品中碳水化合物质量控制研究进展

原料乳与乳制品中碳水化合物具有极高的营养价值,对人体健康具有重要作用,但其掺伪现象较普遍,严重影响了原料乳与乳制品的品质。文章综述了原料乳与乳制品中碳水化合物及其掺伪物质检测技术研究进展,旨在为做好原料乳与乳制品中碳水化合物质量控制提供科学依据。      

Enterotoxigenicity and drug sensitivity of Aeromonas hydrophila isolated from well water in Sweden: a case study

A large number of Aeromonas spp. have been found in drinking water from a drilled well in Sweden. Isolates identified as A. hydrophila were tested for production of enterotoxin, hemolysin, enzymes and for resistant patterns to different antibiotics. The enterotoxin-producing A. hydrophila could be responsible for the long-term diarrhoeal case of a 1 1/2 year old child who consumed the contaminated water.