Synergistic effect between Helichrysum italicum essential oil and cold nitrogen plasma against Staphylococcus aureus biofilms on different food‐contact surfaces

Staphylococcus aureus is the most common pathogen in human, and the most diseases produced by S. aureus are associated with biofilms. Helichrysum italicum essential oil (EO), as a natural and safe spice, was employed to disperse S. aureus biofilm by cold nitrogen plasma (CNP) assist. After S. aureus biofilm formation on food container surfaces, they were exposed to CNP and then treated with Helichrysum italicumEO for biofilm dispersion. The population of S. aureus biofilm was approximately reduced by 2 logs after individual treatment of 0.5 mg mL^?1 Helichrysum italicumEO or 400 W CNP. Interestingly, the combined treatment of Helichrysum italicumEO (0.5 mg mL^?1, 40 min) and CNP (400 W, 1 min) reduced the S. aureus viable count in biofilm below 2 logs CFU per cm² after 1‐day storage. Therefore, the synergetic treatment holds great promise to improve the current treatment systems of bacterial contamination on different food‐contact surfaces.     

The prevention and removal of biofilm formation of Staphylococcus aureus strains isolated from raw milk samples by citric acid treatments

In this study, the antibiofilm activity of citric acid treatment on Staphylococcus aureus strains isolated from raw milk samples was evaluated. For this purpose, the prevention and removal of biofilm formation of S. aureus strains by citric acid treatments (2% and 10%) for 20 min were investigated for comparison with peracetic acid treatment (0.3%) on both microtitration plate and stainless steel coupons. The results indicated that the prevention and removal of biofilm formation and the numbers of prevented or removed S. aureus strains using citric acid treatments were observed to be higher than those using peracetic acid treatment on both surfaces. The prevention and removal of biofilm formation were substantially higher when the concentration of citric acid treatment increased from 2% to 10% and the stainless coupons were used. The results show that citric acid can be used as an alternative disinfectant in controlling biofilm formation in the dairy industry.     

Volatile organic compounds associated with milk spoilage by psychrotrophic bacteria

The volatile organic compounds of milk contaminated with psychrotrophic bacteria were studied by HS‐SPME and GC/MS. Pseudomonas fluorescens PS14, Pseudomonas fragi PS55, Pseudomonas mosselii PS39, Pseudomonas rhodesiae PS62 and Serratia marcescens S92 were inoculated in sterilised milk (2.5% fat) stored at either 5 °C or 10 °C. A total of 47 volatile organic compounds (VOCs) belonging to seven chemical groups were identified in the spoiled milk. Volatile organic compound patterns peculiar to the inoculate bacterial strains were highlighted. 3‐Methylbutan‐1‐ol, 2 methylpropan‐1‐ol, 3‐hydroxybutan‐2‐one, butan‐2,3‐dione, butanoic and hexanoic acids were revealed as potential chemical spoilage indexes of milk spoilage due to the activity of the five psychrotrophic strains studied.     

Formation and dispersal of biofilms in dairy substrates

The formation and dispersion of biofilms in the dairy industry is a problem because it increases cross‐contamination, affecting the shelf life of the products and their safety. The objective of this study was to evaluate the influence of different dairy substrates (cows’ milk and whey protein) on the formation and dispersion of Enterococcus faecalis, Listeria monocytogenes, Staphylococcus aureus and Bacillus cereus in two biofilm systems (mono‐species and multi‐species) on stainless steel at 25 °C. The dominant behaviour of E. faecalis occurred in most of the tests on mono‐species and multi‐species biofilms. A greater dispersion of biofilm cells was observed in skimmed milk.     

Characterisation of Staphylococcus aureus strains from milk and goat cheese and evaluation of their inhibition by gallic acid,nisin and velame of the Brazilian caatinga

Twenty isolates from milk and goat cheese were confirmed as Staphylococcus aureus. These isolates were characterised for phenotypic properties related to cell adhesion and for the presence of enterotoxin production, intercellular adhesion and β‐lactam resistance genes. Staphylococcus aureus L47 showed cell adhesion ability and positivity for the sec, sed, icaD, mecA and blaZ genes. Three antimicrobial compounds were tested singly or in pairs for growth control of strain L47: gallic acid (GA), nisin and essential oil (EO) of Croton heliotropiifolius (velame). At 24 h, EO and EO + nisin showed higher inhibitory activity against S. aureus L47 in goat milk.     

Effect of nisin‐producing Lactococcus lactis starter cultures on the inhibition of two pathogens in ripened cheeses

The effect of Lactococcus lactis nisin‐producing strains, isolated from Italian fermented foods, on the survival of two foodborne pathogens namely Listeria monocytogenes and Staphylococcus aureus was investigated in experimental cheese production. One of the three Lactobacillus lactis nisin innoculated as starters, Lactobacillus lactis 41FL1 lowered S. aureus count by 1.73 log colony‐forming units (cfu)/g within the first 3 days, reaching the highest reduction, 3.54 log cfu/g, by the end of ripening period of 60 days. There was no effect on L. monocytogenes. The application of L. lactis 41FL1 as bioprotective culture in controlling S. aureus shows considerable promise.     

Occurrence and antibiotic resistance of Escherichia coli,Staphylococcus aureus and Bacillus cereus in raw milk and dairy products in Turkey

In this survey, 150 samples of raw milk, white cheese and ice cream from three different dairy‐processing plants in Ankara were analysed to find out if they were contaminated with Escherichia coli, Staphylococcus aureus or Bacillus cereus. The highest contamination percentages were found in raw milk samples as follows: B. cereus (90%), E. coli (74%) and S. aureus (56%) followed by cheese (70% B. cereus, 60% E. coli, and 48% S. aureus) and ice cream (56% E. coli, 36% S. aureus and 20% B. cereus). The survey showed that 2% of cheese samples were contaminated with E. coli O157. It was also found that the numbers of S. aureus and E. coli in raw milk, cheese and ice cream samples exceeded the numbers permitted under the Turkish Food Codex (TFC). The number of B. cereus in raw milk, cheese and ice cream samples was lower than the limit given in the TFC standards. The study also showed that E. coli and S. aureus exhibit resistance to ampicillin, penicillin, tetracycline, erythromycin, gentamicin and trimethoprim/sulfamethoxazole. Escherichia coli isolates also showed resistance to chloramphenicol and ciprofloxacin but none of them exhibited resistance to cefotaxime. All S. aureus isolates were found to be susceptible to cefotaxime, chloramphenicol, and ciprofloxacin. Bacillus cereus isolates were found to be resistant to ampicillin, penicillin and trimethoprim/sulfamethoxazole and sensitive to cefotaxime, chloramphenicol, ciprofloxacin erythromycin, gentamicin and tetracycline.     

3‐Hydroxypyridinone‐l‐phenylalanine conjugates with antimicrobial and tyrosinase inhibitory activities as potential shrimp preservatives

To explore the potential of novel tyrosinase inhibitors, 3‐hydroxypyridinone‐l ‐phenylalanine conjugates, as shrimp preservatives, compounds 1 and 2 were evaluated for the antimicrobial activity, and compound 2 was investigated for the shrimp preservative efficacy. It was found that they both possess a stronger antibacterial effect than kojic acid against two Gram‐positive bacteria and three Gram‐negative bacteria. The minimum inhibitory concentrations (MICs) of compound 2 against Escherichia coli, Staphylococcous aureus, Salmonella gallinarum, Vibrio parahaemolyticus and Bacillus subtilis were determined as 18.5, 37, 148, 37 and 295 μg mL^?1, respectively, whereas MICs of kojic acid against the same 5 bacterial strains were determined to be 355, 178, 1420, 1420 and 355 μg mL^?1, respectively. It has also been demonstrated that treatment with compound 2 improves the sensory properties, retards the growth of spoilage bacteria, decreases the total volatile basic nitrogen (TVB‐N) and increases the pH of Penaeus vannamei Boone, thereby extending the shelf life to 10 days. In contrast, the shelf life of shrimp treated with kojic acid and the control group was 7 and 6 days, respectively. Clearly, 3‐hydroxypyridinone‐l ‐phenylalanine conjugates could find application as shrimp preservatives by inhibiting melanosis and by preventing the growth of bacteria during the storage.     

Determination of Enterotoxigenic and Methicillin Resistant Staphylococcus aureus in Ice Cream

The aim of this study was to determine the prevalence of enterotoxigenic and methicillin‐resistant Staphylococcus aureus in ice creams. After culture‐based identification of isolates, the presence of 16S rRNA and nuc was confirmed by mPCR. S. aureus was identified in 18 of 56 fruity (32.1%), 4 of 32 vanilla (12.5%), and 1 of 12 chocolate (8.3%) ice creams. S. aureus was identified as 38 isolates in 23 ice cream samples by culture‐based techniques, but only 35 isolates were confirmed by PCR as S. aureus. To determine the enterotoxigenic properties of PCR‐confirmed S. aureus isolates, a toxin detection kit was used (SET RPLA®). Of the 12 enterotoxigenic S. aureus isolates, 9 SEB (75%), 1 SED (8.3%), 1 SEB+SED (8.3%), and 1 SEA+SEB+SED (8.3%) expressing isolates were found. The presence of enterotoxin genes (sea, seb, sed) was identified in 13 (37.1%) out of 35 isolates by the mPCR technique. In the ice cream isolates, the sea, seb, and sed genes were detected: 1 sea (7.6%), 9 seb (69.2%), 1 sed (7.6%), 1 seb+sed (7.6%), and 1 sea+seb+sed (7.6%), respectively. The sec gene was not detected in any of these isolates. One of the 35 (2.8%) S. aureus strain was mecA positive.     

Profile of antioxidant and antibacterial activities of different solvent extracts from Rabdosia rubescens

The objective of this work was to investigate the antioxidant and antibacterial activities of different extracts from Rabdosia rubescens and to further evaluate the antibacterial mechanism of extracts. The results showed that 80% acetone extracts had the highest contents of total polyphenols (8.09 mg GAE g^?1) and flavonoids (5.69 mg RE g^?1) and exhibited the strongest antioxidant activities, followed by 80% methanol and 80% ethanol, and the lowest for hexane extracts. Others except for hexane extracts showed different antibacterial activities against Gram‐positive strains, while no inhibitory effects were found on tested Gram‐negative bacterial strains. Among these extracts, 80% acetone and ethanol extracts had relatively higher antibacterial activities with the lowest minimum inhibitory concentration and minimum bactericidal concentration values of 5 and 10 mg mL^?1. The antibacterial mechanism of ethanol extracts against Staphylococcus aureus might be described as it disrupts cell wall, increases cell membrane permeability and then leads to the leakage of cell constituents.     

A survey of the occurrence and properties of methicillin‐resistant Staphylococcus aureus and methicillin‐resistant Staphylococcus intermedius in water buffalo milk and dairy products in Turkey

Antimicrobial resistance, β‐lactamase activity and mecA gene of Staphylococcus aureus and Staphylococcus intermedius isolated from raw water buffalo milk and dairy products in Turkey were determined. All strains showed resistance to at least one antibiotic but none was resistant to vancomycin. Of the 97 S. aureus and 35 S. intermedius strains, 9 (9.2%) and 2 (5.7%) were resistant to oxacillin and harboured mecA gene. β‐lactamase activity of 13.4% and 5.7% of S. aureus and S. intermedius strains was positive, respectively. Overall, 2.5% and 0.55% of the samples were contaminated with methicillin‐resistant S. aureus and S. intermedius, respectively.     

Antibacterial and antioxidant properties of <Emphasis Type="Italic">Ramulus Cinnamomi</Emphasis> using supercritical CO<Subscript>2</Subscript> extraction

In this study, crude extracts of Ramulus Cinnamomi from supercritical carbon dioxide under various extraction conditions were examined for their antioxidant and antibacterial activity. The extractions were conducted in the range of 4,000–6,000 psi and 40–50 °C, and the solvent to feed ratio of the extraction was 30. The antibacterial activity was tested on the clinical drug-resistant strains, including 27 Acinetobacter baumannii, 20 Pseudomonas aeruginosa and 2 Staphylococcus aureus isolates by the disk diffusion method. The bioassay results indicated that Ramulus Cinnamomi showed obvious antimicrobial activity against the tested strains. This study also found that increasing the temperature and pressure would increase the yield of the supercritical fluid extraction (SFE), even though the best extraction conditions for antibacterial activity were found to be high pressure and low temperature. The minimum inhibitory concentration (MIC) was determined on the crude extract of Ramulus Cinnamomi, indicating that the crude extracts from supercritical extraction showed better antibacterial activity than those obtained by ethanol extraction. Based on the spectrophotometer and bioassay determination, the antimicrobial constituent was identified to be cinnamaldehyde. Time-kill kinetics and scanning electron microscopy (SEM) were employed to monitor the survival characteristics and the changes in morphologies, respectively, of the test microorganisms in the presence of herbal extracts. Moreover, antioxidant activity was evaluated by scavenging of the free radical DPPH. Extracts of Ramulus Cinnamomi provided 50% inhibition at 2 mg/ml concentration. This study will provide valuable information for extraction of the natural bioactive component, cinnamaldehyde, from Ramulus Cinnamomi by supercritical extraction.     

Detection of Salmonella spp., Yersinia enterocolitica,Listeria monocytogenes and Campylobacter spp. by real‐time multiplex PCR using amplicon DNA melting analysis and probe‐based assay

Syto9 and probe‐based multiplex real‐time PCR assays for simultaneous detection of a group of foodborne pathogens (named SYLC group), targeting Salmonella spp. (invA gene), Yersinia enterocolitica (ystA gene), Listeria monocytogenes (hly gene) and Campylobacter spp. (rrna gene), have been developed. The Syto9 assay generates amplicon DNA melting curve with four peaks of 86.5 ± 0.5, 84 ± 0.5, 81.5 ± 0.5 and 90.5 ± 0.5 °C corresponding Salmonella spp., Y. enterocolitica, L. monocytogenes and Campylobacter spp. targets, respectively. The sensitivities of the Syto9 and TaqMan assays in artificially inoculated chicken wing rinses were in a range of 3.2 × 10² to 3.1 × 10⁴ and 9.8 × 10² to 1.9 × 10⁴ colony‐forming units per millilitre, respectively, depending on the pathogen. All tested target strains (n = 100) were correctly detected by the both assays, whereas nontarget strains (n = 100) demonstrated no cross‐reactivity representing 100% specificity. The assays are suitable for application in qualitative and quantitative detection of SYLC group pathogens in food matrices.     

Analysis of volatile compounds in L. edodes blanched by hot water and microwave

The comprehensive flavour characterisation and volatile compounds of raw L. edodes, hot water blanching (HB) sample and microwave blanching (MB) sample were comparatively analysed by electronic nose technology and headspace solid‐phase micro‐extraction combined with gas chromatography‐mass spectrometry (HS‐SPME‐GC‐MS). Results indicated that volatile components in L. edodes markedly changed after HB and MB. Volatile compounds of raw L. edodes consisted mainly ketones, sulphide and alcohols, and 3‐octanone, as well as 1‐octen‐3‐one, were the major compounds. The content of ketones and sulphides in blanched samples markedly decreased, while the relative content of aldehydes, hydrocarbons and esters increased, which became the major volatile compounds of treatment samples. In addition, the percentage contents of esters, alcohols and sulphides in MB samples were significantly (P < 0.05) higher than that in HB samples, especially 1‐octen‐3‐ol, which contributes more to mushroom flavour. Therefore, MB is a better pretreatment method of L. edodes processing and cooking according to the results of experiment.     

Antioxidant activities of volatile components isolated from Eucalyptus species

Extracts of three species of eucalyptus leaves (Eucalyptus polyanthemos Schauer, E globulus Labill and E perriniana) and their major volatile components were assessed for antioxidant activity in two different assays. The inhibitory effect of each extract and its components towards aldehyde/carboxylic acid conversion was measured over a period of 30 days. Their inhibitory effects towards malonaldehyde formation from lipid by oxidation with Fenton's reagent were also measured; the extract from E polyanthemos inhibited the oxidation of hexanal completely for 30 days at a level of 200 and 500 µg ml^?1. It also inhibited malonaldehyde (MA) formation in cod liver oil by 86% at the 160 µg ml^?1 level. At the 500 µg ml^?1 level, thymol, 1,8‐cineole, benzyl alcohol and terpinen‐4‐ol identified in the extract from E polyanthemos reduced the extent of hexanal oxidation over a period of 30 days by 100, 96, 82 and 75% respectively. In the lipid/MA assay, thymol, benzyl alcohol, terpinen‐4‐ol and 1,8‐cineole inhibited MA formation by 80, 63, 58 and 26% respectively at the level of 160 µg ml^?1. Thymol exhibited potent antioxidant activity comparable to that of the known natural antioxidant α‐tocopherol. © 2001 Society of Chemical Industry     

Selection of Streptococcus thermophilus strains able to produce exopolysaccharides in milk

A collection of 139 Streptococcus thermophilus strains was genetically screened by PCR targeting some genes involved in exopolysaccharide (EPS) biosynthesis. Twenty‐nine strains were found to be PCR positive and were grown in skim milk to highlight their ropy character, that is the ability to efficiently thicken the milk cultures and minimise syneresis. Three strains producing a skim milk culture with apparent viscosities of 275 ± 14, 340 ± 21 and 510 ± 42 mPa.s, significantly higher than the average value (135 ± 87.3 mPa.s), were selected. EPS from milk cultures of these three strains was extracted and quantified from 84.4 to 209.2 mg/L.     

First report of methicillin‐resistant Staphylococcus aureus in tank cultured dusky kob (Argyrosomus japonicus), and evaluation of three phenotypic methods in the detection of MRSA

This study was conducted to ascertain the occurrence of methicillin‐resistant Staphylococcus aureus (MRSA) in marine finfish (Argyrosomus japonicus) harvested from the wild and two re‐circulatory aquaculture systems, and evaluating the reliability of three phenotypic methods in the detection of methicillin resistance. A total of 120 dusky kob fish were sampled for S. aureus detection using conventional methods and confirmed by polymerase chain reaction (PCR) targeting the nuc gene. Methicillin resistance was determined by molecular detection of the mecA gene. Using mecA as the defining standard, the specificities and sensitivities of cefoxitin disc diffusion, oxacillin screen agar, and growth of S. aureus on Brilliance MRSA II Agar were evaluated. A total of 321 presumptive S. aureus isolates were recovered by culture, out of which 202 (62.9%) were confirmed by PCR. Of these, 33 (16.3%) strains were mecA positive while 169 (83.7%) were mecA negative. The sensitivities and specificities of MRSA detection was 93.9 and 91.7%, 81.8 and 92.3%, and 87.9 and 94.1% for cefoxitin disc, oxacillin screen agar test, and Brilliance MRSA II agar, respectively. This is so far the first report of MRSA in dusky kob aquaculture in South Africa. In the absence of molecular techniques, cefoxitin disc diffusion test is recommended along with any other phenotypic method to improve MRSA detection from samples of veterinary origin.

Practical implications

Methicillin‐resistant Staphylococcus aureus currently presents one of the greatest challenges for medical research worldwide, as well as is one of the most important causes of bacteria gastroenteritis due to preformed toxins in foods. There is a dearth of knowledge on marine foods as carriers/sources of MRSA infection. There are also major discrepancies obtained with MRSA detection methods, making effective detection of this pathogen complicated. The results from this study show that healthy aquaculture fish are reservoirs of MRSA, thus it is necessary to regularly monitor marine foods. Cefoxitin disc diffusion test is recommended as the preferred method for detection of MRSA from fish/food samples where molecular methods are lacking.     

Determination of the relationship between oxalate degradation and exopolysaccharide production by different Lactobacillus probiotic strains

The aim of this study was to evaluate the protective effects of exopolysaccharide (EPS) production on resistance to gastrointestinal digestive conditions and oxalate by probiotic strains of the species Lactobacillus rhamnosus, Lactobacillus fermentum and Lactobacillus brevis. The oxalate‐degrading ability of the strains was determined using an enzymatic assay. The correlation between oxalate degradation rate and EPS production was not significant (P > 0.05); however, the high‐EPS‐producing L. fermentumIP5 and L. brevisYG7 strains showed high oxalate‐degrading activity, whereas the low‐EPS‐producing strain L. fermentumBP5 demonstrated low oxalate‐degrading activity. The present findings suggest that dietary supplementation with the probiotic strain L. fermentumIP5 could be a promising strategy for the prevention of oxalate stone disease.     

Selection and identification of protective culture for controlling Staphylococcus aureus in fresh Domiati like cheese

Antibacterial activity of forty lactic acid bacteria (LAB) isolates toward Staphylococcus aureus was evaluated. The selected strains were then used as protective culture in artificial contaminated Domiati like cheese with S. aureus. The effect of using these strains on physicochemical properties and overall acceptability of fresh cheese was evaluated. Depending on its antibacterial activity, three strains of Lactobacillus rhamnosus 130RZFAAU, 131RZFAAU, and 190RZFAAU were selected for cheese making. No negative sensory properties were observed by the panelists when LAB strains were used as a single culture in the fresh cheese making. The application of these strains as protective culture in artificial contaminated cheesemaking process give a positive results. S. aureus was detected in cheese samples by culture method and propidium mono azide–quantitative polymerase chain reaction method. The results recommended that the strain L. rahmnosus 131RZFAUU that used in this study has antimicrobial activity against S. aureus and could be used as protective culture for improving the safety of Egyptian soft cheese.

Practical applications

Detection of pathogenic bacteria by classical tests can take several days. It would be useful to have a rapid detection protocol to screen for the presence of Staphylococcus aureus in milk and cheese. Application of real‐time PCR in cheese is sufficient in characterization the S. aureus communities in raw milk and follow the dynamics of the entire populations in cheese. Recently, some scientific publications have shown that the naturally cheese microflora can efficiently prevent the growth of pathogenic or spoilage microorganisms. The control of spoilage and pathogens bacteria has been traditionally done by chemical additives, but the application of promising protective cultures, especially for traditionally cheeses made from raw milk, is limited. This work present some protective culture selected for controlling S. aureus in soft cheese. This work confirm the PMA‐q PCR method for detection live cells of S. aureus in cheese rapidly.     

Short communication: Methicillin-resistant Staphylococcus aureus detection in US bulk tank milk

Staphylococcus aureus is a major cause of mastitis in dairy cattle. This study estimated the herd prevalence of methicillin-resistant Staph. aureus (MRSA) among US dairy herds by testing bulk tank milk (BTM) samples using genotypic and phenotypic methods. A nationally representative sample of 542 operations had BTM cultured for Staph. aureus, and 218 BTM samples were positive upon initial culture. After 4 wk to 4 mo of frozen storage, 87% of 218 samples (n = 190) were still culture positive for Staph. aureus on blood agar, but none were positive for MRSA on the selective indicator medium CHROMagar MRSA. A duplex PCR was used to detect the Staph. aureus-specific nuc gene and the methicillin resistance gene, mecA, in mixed staphylococcal isolates from the 190 BTM samples that were positive for Staph. aureus after storage. Seven samples tested positive for nuc and mecA, and 2 samples tested positive for mecA only. MecA-positive Staphylococcus spp., but not MRSA, were subsequently isolated from 5 samples, whereas neither mecA-positive Staphylococcus spp. nor MRSA was isolated from the remaining 4 samples. Presence of methicillin-resistant, coagulase-negative Staphylococcus spp. may complicate the detection of MRSA by means of PCR on BTM. Bulk tank milk in the United States is not a common source of MRSA.