Effect of Zataria multiflora Boiss. essential oil and nisin on Salmonella typhimurium and Staphylococcus aureus in a food model system and on the bacterial cell membranes

The effects of different concentrations of Zataria multiflora Boiss. essential oil (EO: 0, 5, 15 and 30 μl 100 ml⁻¹) and nisin (N: 0, 0.25 and 0.5 μg ml⁻¹), temperatures (T: 25 and 8 °C), and storage times (up to 21 days) on growth of Salmonella typhimurium and Staphylococcus aureus in a commercial barley soup were evaluated in a factorial design study. The growth of S. typhimurium was significantly (P < 0.05) decreased by EO concentrations and their combinations with N concentrations at 8 °C. For S. aureus, the viable count was significantly (P < 0.05) inhibited by EO and N concentrations and their combinations, incubated at both storage temperatures. The mechanism of the antimicrobial action of EO, N, and their combinations against cell membranes of the tested organisms were also studied by measurement of the release of cell constituents and by the electronic microscopy observations of the cells. The significant increase of the cell constituents’ release of both organisms was observed as a result of treatments with EO and EO in combination with N. Electronic microscopy observations revealed that the cell membranes of S. typhimurium treated by EO and EO in combination with N were significantly damaged, while cells treated with only N looked similar to untreated cells. The electron micrographs of treated cells of S. aureus with EO, N, and their combination also showed important morphological damages and disrupted membranes.     

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.     

Enhanced antimicrobial activity of nisin in combination with allyl isothiocyanate against Listeria monocytogenes,Staphylococcus aureus,Salmonella Typhimurium and Shigella boydii

This study was designed to evaluate the synergistic antimicrobial effect of nisin and allyl isothiocyanate (AITC) against Listeria monocytogenes, Staphylococcus aureus, Salmonella Typhimurium and Shigella boydii. The synergistic interactions between nisin and AITC were observed against all foodborne pathogens, showing the fractional inhibitory concentrations <1. The populations of L. monocytogenes and S. aureus at the combined treatment of nisin and AITC were decreased to below 1 log CFU mL^?1 after 10‐h incubation at 37 °C. The changes in fatty acid profiles of all strains were substantially influenced by nisin alone and the combined treatment of nisin and AITC. A good agreement was observed among cell viability, membrane permeability and depolarisation activity in response to nisin and AITC. The results suggest that nisin and AITC as synergistic inhibitors could be an effective approach to achieve satisfactory antimicrobial activity against a wide range of foodborne pathogens.     

Application of enterocins A and B, sakacin K and nisin to extend the safe shelf-life of pressurized ready-to-eat meat products

The efficiency of food preservation systems is determined by the technologies that are combined, the intrinsic properties of the food products and the target microorganisms. In the present study, the bacteriocins nisin, enterocins A and B and sakacin K were applied to cooked and dry cured ham spiked with Listeria monocytogenes, Salmonella enterica and Staphylococcus aureus and submitted to a high pressure treatment of 600 MPa. Before pressurization nisin produced significant reductions to the counts of L. monocytogenes and S. aureus, especially in dry cured ham. After the pressurization, Salmonella and L. monocytogenes were not detected in 25 g of both cooked and dry cured ham and remained at this level during the entire storage (57 days at 4 °C + 63 days at 15 °C). S. aureus levels, in contrast, only decreased below the detection limit (1 log CFU/g) in the nisin batches. Afterward, when storage was performed at an abusive temperature, the ability of S. aureus to grow was dependant on the bacteriocin applied and the kind of meat product. Thus, at the end of storage, while S. aureus counts were <1 log CFU/g in all dry cured ham batches, only nisin could inhibit its growth in cooked ham.     

Comparison of the antipathogenic effect toward Staphylococcus aureus of N-linked and free oligosaccharides derived from human,bovine, and goat milk

N-linked oligosaccharides (N-glycans) derived from milk were recently found to be antipathogenic. This study compares the antimicrobial activity of N-linked glycans and free oligosaccharides from human, bovine, and goat milk against Staphylococcus aureus. Milk N-glycans showed a bactericidal/bacteriostatic effect on the pathogen when compared to free milk oligosaccharides, evidenced by the clear zone from the halo assay, with the order of human milk >goat milk >bovine milk. None of the free milk oligosaccharide samples were bactericidal/bacteriostatic, despite its positive results in growth curve and minimum inhibitory concentration (MIC) assays which are believed to be related to hyperosmosis. Both N-glycans and free milk oligosaccharides can reduce the adhesion of Staphylococcus aureus to Caco-2 cells, however, N-glycans worked significantly more effective than free milk oligosaccharides. Structural analysis of all free oligosaccharide and N-glycan samples showed the obvious interspecies differences, and the structure/function relationship of the respected N-glycans is of interest for future study. The significant bactericidal/bacteriostatic activity possessed by human, bovine, and goat milk N-linked glycans holds great potential as a novel substitute for antibiotics.     

Effect of Mentha spicata L. and Mentha aquatica L. essential oils on the microbiological properties of fermented dairy product,kashk

The antibacterial activity of Mentha spicata and Mentha aquatica essential oils (EO) was tested against Staphylococcus aureus, Lactobacillus reuteri, Bifidobacterium animalis and Clostridium perfringens using agar well and disc diffusion techniques. Results showed that M. spicata EO had the highest inhibition activity against the studied microorganisms. Then, the antibacterial activity of both EO at 1500 and 2500 ppm was examined in industrial liquid kashk during the storage at 4 °C for 20 days. Both EO reduced the S. aureus viable count below 5 log CFU g^?1 after 4 days; however, the population of C. perfringens, L. reuteri and B. animalis decreased <1 log CFU g^?1 during the storage time. The least deteriorative effect on the lactic acid bacteria was related to M. aquatica. As revealed by organoleptic studies, kashk samples containing M. aquatica EO at 1500 and 2500 ppm were the most preferred samples.     

Antibiotic Resistance Traits and Molecular Subtyping of Staphylococcus aureus Isolated from Raw Sheep Milk Cheese

The main objective of the present research was to evaluate the antibiotic resistance profiles of Staphylococcus aureus isolated from raw sheep milk cheese. A total of 150 strains were isolated from curd cheese samples and identified as S. aureus. The survey on antibiotic resistance was carried out on 47 strains, selected among isolates showing differences in the banding pattern after Pulsed Field Gel Electrophoresis (PFGE) screening or, belonging at the same pulsotype but isolated from different cheese samples. On selected strains antimicrobial resistance against ampicillin, penicillin, cloxacillin, tetracycline, erythromycin, and vancomycin was assessed by broth microdilution method. The presence of the genes coding for antibiotic resistance and virulence factors (agr alleles, sea‐see, and tst) was also investigated by PCR. Thirty‐one isolates belonging to agrI and agrIII groups carried at least one gene coding for enterotoxins or toxic shock syndrome toxin. Approximately 60% of the selected strains were susceptible to the tested antibiotics. Twelve of 47 isolates showed multiple resistance against ampicillin and penicillin. Only 1 strain, represented by a unique PFGE profile showed simultaneous resistance to ampicillin, penicillin and cloxacillin. Single resistance against tetracycline was found in 5 isolates belonging to 2 different pulsotypes. The results of this study suggest that the recovery of S. aureus resistant strains in raw milk cheese samples is quite common but it is limited to few antibiotic classes, mainly β‐lactams and tetracyclines. None of the strains showed resistance to erythromycin and vancomycin.     

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.     

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.     

Occurrence of foodborne pathogens and characterization of Staphylococcus aureus in cheese produced on farm-dairies

The objective of this study was to address knowledge gaps identified in an earlier risk assessment of Staphylococcus aureus and raw milk cheese. A survey of fresh and short-time ripened cheeses produced on farm-dairies in Sweden was conducted to investigate the occurrence and levels of S. aureus, Listeria monocytogenes and Escherichia coli, to characterize S. aureus isolates with special emphasis on enterotoxin genes, antibiotic resistance, bio-typing and genetic variation, and to collect information related to production practices. In general, the hygienic quality of farm-dairy cheeses appeared to be of an acceptable microbiological quality, e.g. L. monocytogenes and staphylococcal enterotoxin were not detected in cheese samples. However, E. coli and enterotoxigenic S. aureus were frequently found in raw milk cheeses and sometimes at levels that are of concern, especially in fresh cheese. Interestingly, levels in raw milk fresh cheese were significantly lower when starter cultures were used. Up to five S. aureus colonies per cheese, if possible, were characterized and about 70% of isolates carried one or more enterotoxin genes, most common were sec and sea. The Ovine biotype (73%) was most common among isolates from goat milk cheese and the Human biotype (60%) from cow milk cheese. Of all isolates, 39% showed decreased susceptibility to penicillin, but the proportion of isolates from cows' cheese (66%) compared to isolates from goats' cheese (27%) was significantly higher. S. aureus isolates with different properties were detected in cheese from the same farm and, sometimes even the same cheese. Isolates with the same pulsed-field gel electrophoresis (PFGE)-pattern were detected on geographically distant dairies. This indicates that multiple sources and routes of contamination are important. To improve the safety of these products efforts to raise awareness of the importance of hygiene barriers and raw milk quality as well as improved process control can be suggested, e.g. use of starter cultures and monitoring of fermentation with a pH-meter. For future safety assessments, a better understanding of factors determining toxin production in these cheeses is needed.     

Biotyping and enterotoxigenicity of Staphylococci isolated from fresh and frozen meat marketed in Jordan

A total of 286 fresh and processed meat samples marketed in Jordan were collected for isolation and typing of Staphylococci. Devriese’s system was followed for biotyping of the isolated Staphylococcus aureus subsp. aureus and enterotoxigenicity of the isolates was also determined. S. aureus subsp. aureus was found in 80.8% of the samples. Means counts for meats of the various sources ranged from 5.3 × 10² to 4.3 × 10⁴ cfu/g. However, only 33.6% of the samples had coagulase positive Staphylococci. Biotyping of S. aureus subsp. aureus isolates revealed that 12%, 24%, 8%, and 27% are human, bovine, ovine and non-host specific biovars, respectively. Only 28% of these isolates, of unknown type, were isolated from goat’s meat. Enterotoxigenic S. aureus subsp. aureus isolates accounted for 13.5% of a total of 231 Staphylococci. The non-S. aureus enterotoxigenic isolates were identified as Staphylococcus epidermidis, Staphylococcus sciuri and Staphylococcus intermedius. Staphylococci enterotoxin types A, C, and B accounted for 57%, 36% and 7% of the isolated S. aureus subsp. aureus, respectively. This study showed that the enterotoxigenic Staphylococci represent a potential hazard in meat and the Devriese’s biotyping system needs to be extended to accommodate the isolates of unknown type.     

The prevalence,genetic diversity and antibiotic resistance of Staphylococcus aureus in milk,whey, and cheese from artisan farm dairies

In this study, coagulase positive staphylococci were detected in 45% of the 69 bovine milk, whey and cheese samples taken from five farm dairies, and all raw milk samples were contaminated. Genetic diversity, staphylococcal enterotoxin genes and antimicrobial susceptibility in putative Staphylococcus aureus isolates were investigated. Sixty-one percent of the 72 isolates analysed belonged to the same pulsed field gel electrophoresis group. The spa-typing revealed seven different spa types, t2678 being the most prevalent, but t127 and t197 were also detected. Sixteen different toxin gene profiles were identified in 87.5% of the isolates with sec and tst being the most frequent (52.5%), followed by seg and seh. All isolates were methicillin-sensitive S. aureus, and sensitive to the 12 antibiotics tested. The prevalence of S. aureus and the high diversity of isolates carrying enterotoxin genes constitute grounds for food safety concern in artisanal cheese making, whether pasteurised or not.     

Prevalence and Characteristics of Enterotoxin B‐Producing Staphylococcus aureus Isolated from Food Sources: A Particular Cluster of ST188 Strains was Identified

The aim of this study was to investigate the prevalence and characteristics of staphylococcal enterotoxin B (SEB) producing Staphylococcus aureus (S. aureus) isolated from food sources. A total of 412 S. aureus isolates were recovered from 1970 milk and dairy samples (n = 236) and 2450 meat samples (n = 176) in China from 2009 to 2014. Of the 412 isolates, 124 isolates were tested positive for 1 or more classical staphylococcal enterotoxin (SE) genes using PCR, and 31 isolates were positive for seb gene and further proved to be SEB‐producing. Four SE profiles were observed among 31 SEB‐producing isolates when investigated using ELISA kit, that is, SEB (16 isolates), SEA+SEB (6 isolates), SEB+SEC (6 isolates), and SEB+SED (3 isolates). Thirteen sequence types (STs) were identified in the 31 SEB‐producing S. aureus isolates using multilocus sequence typing (MLST). The 3 most detected STs were ST1 (7 isolates), ST188 (6 isolates), ST59 (3 isolates). Two distinct clusters were identified by pulsed‐field gel electrophoresis (PFGE), each of which showed excellent consistency with ST188 and ST1 achieved by MLST, respectively. In summary, this study reveals that various SE profiles are observed in SEB‐producing S. aureus isolates and the great part of SEB‐producing S. aureus isolates are showed as clusters. Especially, a particular cluster of ST188 strains was observed in SEB‐producing S. aureus isolates which was associated with outbreaks of SFP and needs further attention.     

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.     

Essential oil composition and antibacterial activity of the grapefruit (Citrus Paradisi. L) peel essential oils obtained by solvent‐free microwave extraction: comparison with hydrodistillation

The chemical composition of the essential oil (EO) obtained by solvent‐free microwave extraction (SFME) and hydrodistillation (HD) from the peel of grapefruit (Citrus Paradisi. L) was analysed by gas chromatography/mass spectrometry (GC/MS). Totally, twenty‐five components were identified in the EO. Limonene was observed as dominant (91.5–88.6%) for two extraction methods, SFME and HD, respectively. β‐Pinene (0.8–1.2%), linalool (1.1–0.7%), α‐terpinene (0.7–1.0%) and the other minor components were also detected. Disc diffusion method was applied to determine the antibacterial properties of the EO. The results showed that the EO of grapefruit peel had a wide spectrum of antimicrobial activities against Staphylococcus aureus, Enterococcus faecalis, Staphylococcus epidermidis, Escherichia coli, Salmonella typhimurium, Serratia marcescens and Proteus vulgaris, with their inhibition zones ranging from 11 to 53 mm.     

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.     

Short communication: Diversity of species and transmission of antimicrobial resistance among Staphylococcus spp. isolated from goat milk

The increasing production of goat milk and its derivatives is affected by the occurrence of intramammary infections, which are highly associated with the presence of Staphylococcus species, including some with zoonotic potential. Staphylococci in general can exchange mobile genetic elements, a process that may be facilitated by the isolate's capacity of forming biofilms. In this study we identified, to the species level, Staphylococcus isolated from goat milk samples by MALDI-TOF and confirmed the identification by sequencing housekeeping genes (rrs and tuf). Eight species were identified, more than half being either Staphylococcus epidermidis or Staphylococcus lugdunensis. The isolates were shown by pulsed-field gel electrophoresis to be genetically diverse between the studied herds. Resistance to ampicillin and penicillin was widespread, and 2 Staph. epidermidis isolates contained the methicillin-resistance gene mecA. Most of the isolates that were resistant to at least 1 of the 13 antimicrobials tested harbored plasmids, one of which was demonstrated to be conjugative, being transferred from a Staph. epidermidis to a Staphylococcus aureus strain. Biofilm formation was observed in almost every isolate, which may contribute to their capacity of exchanging antimicrobial resistance genes in addition to acting as a physical barrier to the access of drugs. Our results showed that antimicrobial resistance among goat staphylococci may be emerging in a process facilitated by the exchange of mobile genetic elements between the bacteria and the establishment of biofilms, which calls for careful monitoring and more effective control therapies.     

The Use of Multiplex PCR to Determine the Prevalence of Enterotoxigenic Staphylococcus aureus Isolated from Raw Milk,Feta Cheese,and Hand Swabs

Staphylococcus aureus (S. aureus) can cause mastitis in cattle and, therefore, can be present in milk. This study was undertaken to determine the prevalence of coagulase positive S. aureus and its enterotoxin genes sea, seb, and sec in isolates recovered from raw milk, feta cheese, and human hand swabs of milk and cheese handlers in Beni‐Suef province, Egypt. A total of 100 samples of raw milk and 50 samples of pasteurized‐milk feta cheese were collected. In addition, 50 hand swabs from milk handlers and 25 hand swabs from cheese handlers were examined for the presence of coagulase positive S. aureus. The isolates were characterized by multiplex PCR for detection of sea, seb, and sec genes, and for resistance to 5 classes of commonly used antibiotics. Twelve (12/100), 12 (6/50), and 17% (13/75) of milk, cheese, and hand swab samples, respectively, were positive for coagulase positive S. aureus. One isolate was obtained from each positive sample (31 isolates), and none contained genes for SEA or SEC production. Twenty‐five percent, 33%, and 31%, respectively, of the isolates contained the genes for SEB, resulting in 3%, 4%, and 5% of samples being positive for toxin producing coagulase positive S. aureus, respectively. At least one isolate was resistant to each of the antibiotics tested. Despite the low potential for SEB production shown, preventative measures, such as maintenance of the cold‐chain and good hygienic practices should be implemented to further reduce the potential risk to public health from SEB, and to reduce the spread of antimicrobial resistance.