Prevalence and antimicrobial resistance of Listeria species isolated from milk and dairy products in Iran

The objective of this study was to determine the prevalence of Listeria spp. in milk and dairy products in Isfahan province, Iran. From March 2007 to September 2009, a total of 594 samples of various milk and dairy products were obtained from randomly selected retail stores. Using conventional bacteriologic method, 55 samples (9.3%) were positive for Listeria spp. The highest prevalence of Listeria was found in raw sheep milk samples (22.6%), followed by cheese samples (18.9%). The most species recovered was Listeria innocua (58.2%); the remaining isolates were Listeria monocytogenes (32.7%) and Listeria seeligari (9.1%). Overall, 54 Listeria isolates (98.2%) were resistant to one or more antimicrobial agents. Resistance to nalidixic acid was the most common finding (96.4%). All Listeria isolates were susceptible to vancomycin. The results of this study indicate the potential risk of infection with Listeria in people consuming raw and unpasteurized milk and dairy products.     

Slime production,DNase activity and antibiotic resistance of Staphylococcus aureus isolated from raw milk,pasteurised milk and ice cream samples

In this study, a total of 180 samples of raw milk, pasteurised milk and ice cream were analysed for the presence of Staphylococcus aureus. There were 110 S. aureus isolated from these samples, which were investigated for DNase activity, slime production and antibiotic resistance. DNase agar that was used to investigate for DNase activity revealed DNase activity in 94.5% of 110 S. aureus. Slime production of S. aureus that was investigated by using Congo Red Agar method revealed slime production in 52.7% of S. aureus. Resistance of S. aureus to different antibiotics was determined by the Kirby–Bauer disc diffusion test. Resistance to penicillin G, methicillin and bacitracin was frequent for S. aureus strains. Few numbers of the strains were resistant to erythromycin. All strains were susceptible to vancomycin, sulbactam–ampicillin, ciprofloxacin and cefaperazone–sulbactam. Slime production positive strains were tested against penicillin G, methicillin and bacitracin. No difference was found between the antibiotic resistance of slime positive and slime negative S. aureus. There was a high rate of resistance for all S. aureus isolates against penicillin G, methicillin and bacitracin.     

Contamination of post-harvest poultry products with multidrug resistant Staphylococcus aureus in Maryland-Washington DC metro area

We investigated the prevalence of Staphylococcus aureus and multidrug including methicillin-resistant S. aureus (MRSA) in poultry retail meat samples from the Maryland-Washington DC metro area. A total of 24 S. aureus isolates were recovered from 96 whole poultry carcass samples and the prevalence of S. aureus were 25.0%, 14.29%, and 33.3% in retail poultry meats collected from farmers markets, organic and conventional retail supermarkets, respectively. Both single and multi-drug resistance isolates were detected in 58.3% (7/12) isolates from conventional retail meat products but none from farmers markets or organic retail meat isolates. Conventional retail meat isolates were found to be resistant to both erythromycin (50.0%) and tetracycline (58.3%). We also detected an MRSA isolate harboring mecA gene in conventional retail meat which showed co-resistance towards erythromycin, tetracycline, and vancomycin.     

Farm to consumption risk assessment for Staphylococcus aureus and staphylococcal enterotoxins in fluid milk in China

The objective of this study was to conduct a risk assessment to determine the food poisoning risk from the consumption of milk in China that might be contaminated by Staphylococcus aureus. Data related to prevalence and concentration of S. aureus in fluid raw milk in China were collected from the literature and used to calculate the initial contamination levels. Two main consumption routes were considered and the results of the Monte Carlo simulation model indicated that the storage temperature in the processing plant and heat processing of milk in the home were the primary factors affecting the S. aureus concentration at the processing plant and the home before consumption, respectively. Other important factors were distribution of log (D)-value's for S. aureus, storage temperature and time on farm, temperature of the thermal treatment of milk, and treatment time at the dairy processing plant. To minimize the risk of milk-borne staphylococcal intoxication in China, the key step appears to be the control of storage conditions during the period after heat treatment and before consumption. The risk assessment model developed in present study provides valuable information for Chinese government and dairy processors to improve milk safety. It also could provide valuable recommendations for Chinese consumer education on safe handling of milk products.     

Detection of methicillin-resistant Staphylococcus aureus in dairy cow farms

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most common pathogens causing nosocomial infections worldwide. Animal-associated MRSA hazard has been recently identified, but less information is currently available regarding MRSA in cattle. The aim of this study was to estimate the presence of MRSA in samples of bulk milk, environmental dust, conjunctival and nasal swabs of workers obtained from thirty dairy cow farms. A total of 200 S. aureus strains were identified using phenotypic and molecular approaches. Three other species (Staphylococcus epidermidis, Staphylococcus xylosus and Staphylococcus saprophyticus) were found. In five S. aureus strains isolated from environmental dust and one S. epidermidis strain derived from human samples, mecA gene was detected showing a specific fragment at 527 bp. Moreover, 66 S. aureus strains were distinguished by susceptibility to 15 antimicrobial agents. The highest resistance profile was ascribed to ampicillin, amoxicillin and penicillin G, both in workers and bulk milk samples. Generally, a multiple resistance to 4 up to 10 antibiotics was detected. S. aureus mecA⁺ strains and S. epidermidis mecA⁺ strain showed multiple resistance to 13 and 11 antibiotics, respectively. The obtained results suggested that the low number of MRSA strains, probably of human origin, was due to the appropriate hygienic practices adopted by the dairy cow farms.     

Adhesion of Staphylococcus aureus on stainless steel treated with three types of milk

Staphylococcus aureus has the ability to adhere and to form biofilm on inert surface such as stainless steel commonly used in food industry. The biofilm formed on the surface of milk processing equipments could be a source of dairy products contamination. This contamination causes a food poisoning. In this paper the S. aureus adhesion on stainless steel treated by three types of milk (ultrahigh-temperature (UHT)-treated milk; UHT skimmed milk, UHT semi-skimmed milk) was investigated.Stainless steel was exposed to three types of milk with a different amount of fat component. Contact angles measurements were used to determine the surface physicochemical properties of substratum treated with the three milk products. The hydrophobicity and electron acceptor properties of stainless steel seem to be decreasing with the amount of fat component present in milk but its electron donor property increase with this component. The ability of S. aureus to adhere to stainless steel treated and untreated with milk was also examined. Treatment with the three types of milk reduces bacterial attachment. On treated substratum, the adhesion extent was affected by the type of milk and consequently by the amount of fat component. The lower and the higher adhesion were obtained when the steel was treated by the UHT semi-skimmed milk and UHT skimmed milk respectively. The correlation between physicochemical properties and S. aureus adhesion show that this latter was controlled by hydrophobicity and electron donor properties.The findings of this work can contribute to develop strategies for prevent S. aureus adhesion on stainless steel and biofilm formation. Also they could be taken into account in cleaning and disinfection procedures.     

Staphylococcus aureus and methicillin-resistant Staphylococcus aureus in retail raw chicken in China

The aim of this study was to determine the prevalence of Staphylococcus aureus (S. aureus) and methicillin-resistant S. aureus (MRSA) in whole raw chickens in China. A total of 1152 whole chicken samples were collected from three types of retail markets (large supermarket, small supermarket, and farmers market) in six provinces and two national cities in China and screened for the presence of. S. aureus, then the S. aureus isolates were further examined for the presence of MRSA. The overall positive rate for S. aureus was 24.2%. S. aureus prevalence was highest in Shaanxi province (39.6%), followed by Fujian province (31.3%), then Sichuan and Guangdong provinces (26.4% in each province), Shanghai (23.6%), Beijing (19.4%), Henan province (13.9%), the lowest prevalence was found in Guangxi province (13.2%). S. aureus prevalence was significantly different among the six provinces and two national cities (P < 0.05). Among 1152 chicken samples, twenty samples (1.7%) were positive for MRSA. MRSA prevalence was highest in Shaanxi province and Sichuan province (3.5% for each), then Fujian province (2.8%), Guangdong province and Shanghai (2.1% for each); MRSA isolates were not detected from Beijing, Henan province and Guangxi province. The overall S. aureus prevalence in chickens collected from large supermarket, small supermarket, and farmers market were 26.3%, 25%, and 21.4%, respectively, but differences were not significant (P > 0.05). These findings indicate the need for identifying the factors contributing to the contamination at the farm, processing and retail level, and for the implementation of good manufacturing practice to better control the pathogen throughout poultry industry.     

Prevalence,genetic diversity and antimicrobial susceptibility of Listeria monocytogenes isolated from open-air food markets in Greece

A total of 210 food samples originating from milk products, ready-to-eat salads, raw meat and raw meat products purchased in ten open-air market places in Thessaloniki, Greece, were analyzed for the presence of Listeria monocytogenes. Thirty (14.3%) contained L. monocytogenes with the highest prevalence in raw meat (27.5%), raw meat products (18%) and cheese (8%). The strains were susceptible to 16 antimicrobials as determined by microbroth dilution, except one strain which displayed resistance to tetracycline (MIC > 32 μg/ml). This strain carried the tetracycline resistance gene tet(M). Pulsed-field gel electrophoresis (PFGE) revealed a low genetic diversity among the isolates, irrespective of their origin. This suggests that dominant L. monocytogenes clones are widespread in different food product types in open-air food markets in Greece. The high prevalence of L. monocytogenes in these products indicates that appropriate hygienic measures and periodic bacteriological controls are also necessary in open-air food markets to reduce contamination with food-borne pathogens. Greek specialties made with raw meat and raw milk may contain L. monocytogenes and should not be consumed by persons at risk.     

Aflatoxin M1 in milk and traditional dairy products from west part of Iran: Occurrence and seasonal variation with an emphasis on risk assessment of human exposure

In the present study, a total of 358 samples consisting of raw milk of cow (n = 64), goat (n = 56) and sheep (n = 52); traditional cheese (n = 40), yoghurt (n = 42), Kashk (n = 40), Doogh (n = 44) and Tarkhineh (n = 20) were analyzed for aflatoxin M₁ (AFM₁) by using an enzyme linked immunosorbent assay (ELISA). Frequency of AFM₁ and its concentration ranges in the ELISA positive samples were determined by high performance liquid chromatography with fluorescence detection (HPLC-FD). AFM₁ contamination was 84.3%, 44.6% and 65.3% for cow, goat and sheep raw milks, respectively. Moreover, AFM₁ was in 65.5%, 23.8%, 14%, 13.6% and 35.0% of cheese, yoghurt, Kashk, Doogh and Tarkhineh samples, respectively. Percentages of cow milk, goat milk, sheep milk and cheese samples exceeding the EU limit were 35.9%, 11.1%, 26.9% and 10%, respectively. HPLC analyses confirmed the ELISA results although the percentages of AFM₁ contamination in raw milk and dairy products were lower than that of ELISA. There were significant differences (P < 0.05) between the mean AFM₁ contents of raw milk, cheese and yoghurt samples during winter and summer seasons. Our study demonstrated that there is a potential risk for liver cancer due to the consumption of milk and dairy products in Iranian consumers.     

Antimicrobial resistance of Escherichia coli and Staphylococcus aureus isolated from food handler's hands

The aim of this study was to determine the antimicrobial resistance of Escherichia coli and Staphylococcus aureus isolates from food handlers' hands at primary schools in Hulu Langat district, Selangor (Malaysia). Disc diffusion methods were used to examine the antimicrobial resistance of the bacteria by using ten types of antibiotic discs with different concentrations. The results show that the prevalence of S. aureus (65.88–74.12%) was far higher than the prevalence of E. coli (9.41–14.12%). The percentage isolates of E. coli that were resistant to the antibiotics was 85.71% Penicillin and Chloramphenicol, 57.14% Sulfamethoxazole-Trimethoprim, Ampicillin and Trimethoprim, 28.57% Kanamycin and Tetracycline and 14.29% Ciprofloxacin. All of the isolates had shown susceptible to Gentamicin and Nitrofurantoin. For S. aureus, the percentage isolates that were resistant to the antibiotics was 72.30% Ampicillin, 53.38% Penicillin, 4.73% Nitrofurantoin,1.35% Chloramphenicol and Trimethoprim and 0.68% Kanamycin and Tetracycline. None of the isolates had shown resistant to Ciprofloxacin, Sulfamethoxazole-Trimethoprim and Gentamicin. Multidrug resistant Escherichia coli represented a high percentage (85.71%) of the total positive strains revived whereas multidrug resistant S. aureus strains were only 5.41% of the total positive strains. The existence of multidrug resistant bacteria is quite worrying as they may pose serious threat to the patients. Hence, the microbiological quality of food handlers' hands from foodservice operations should be maintained in a good condition to reduce the existence of multidrug resistance bacteria.     

Seasonal variation of aflatoxin M1 contamination in industrial and traditional Iranian dairy products

This study aimed to determine the occurrence of aflatoxin M₁ (AFM₁) contamination in 682 dairy product samples consisting of raw milk of cow, goat and sheep; Lighvan cheese; and industrial and traditional yoghurt, Kashk and Doogh samples collected from popular markets and dairy ranches in four large Iranian cities. Thin layer chromatography (TLC) technique was used for analysis of the samples. Results showed that the incidence and levels of AFM₁ contamination in raw cow milk and industrial products (manufactured from cow milk) were higher than raw goat or sheep milk, and traditional products (made from goat and sheep milk), respectively. Moreover, seasonal variations influenced the concentration of AFM₁ in most of the analyzed dairy products. Owing to the abundance and popularity of the industrial products, contamination of these products in such a level could be a potential hazard for public health.     

Listeria spp. in the raw milk and dairy products in Antakya,Turkey

In this study, the presence of Listeria spp. was investigated in a total of 157 raw milk and dairy product samples sold in Antakya (Antioch). The prevalence of Listeria spp. in raw milk and Turkish white cheese samples was found to be 2.12% and 8.23%, respectively. Listeria monocytogenes was not isolated from raw milk and found in only two cheese samples (2.35%). No Listeria spp. were isolated in any of the butter and yoghurt samples.     

Incidence and genetic variability of Listeria species from three milk processing plants

The presence of Listeria in three milk processing environments as a potential source of milk contamination was assessed. Swab samples (n = 210) taken from milk processing plants were examined. Sample sites included the milk processing equipment, besides areas handling raw and pasteurized milk. The USDA Listeria-selective enrichment procedure was used to process the samples. Forty one (19.52%) Listeria isolates were recovered. The isolates were further subjected to biochemical and genotypic characterization. Out of 41 isolates, 16 (7.62%) were confirmed as Listeria monocytogenes, 2 (0.95%) as L. ivanovii, 19 (9.05%) as L. innocua. 1 (0.48%) as L. seeligeri and 3 (1.43%) as L. grayi. All the L. monocytogenes isolates were positive for the hlyA gene. PCR based serotyping revealed all L. monocytogenes to be of 1/2a, 1/2c, 3a and 3c serovar group. AscI and ApaI restriction analysis yielded four PFGE clusters for 16 L. monocytogenes isolates obtained from raw milk collector, milk silos, buttermilk mixer, cheese and other milk product processor. No predominant PFGE cluster was observed among these L. monocytogenes isolates. The main sources of L. monocytogenes were found to be raw milk collector and milk silos. In the present study L. monocytogenes was isolated from milk and milk products processing plants which could cross-contaminate the processed products and may possess a potential threat to public health.     

Contamination of milk with Bacillus cereus by post-pasteurization surface exposure as evaluated by automated ribotyping

Over the last decade, increased transportation of refrigerated raw milk from farms to factories has raised concerns over Bacillus cereus contamination in the Brazilian dairy industry. Twelve isolates from pasteurized milk and 30 isolates from the post-pasteurization equipment surfaces of a dairy processing unit were characterized as B. cereus. Seven ribotypes were identified, demonstrating the genetic variability of this microorganism. Most of the isolates belonged to the same ribogroup (RIBO1222-73-S4), and they were found on four surfaces and also in the milk, indicating the role of the equipment surfaces as reservoirs for milk recontamination.     

The specific antibacterial effect of the Salvia oil nanoliposomes against Staphylococcus aureus biofilms on milk container

The Staphylococcus aureus (S. aureus) biofilm on container is the main source of microbial contamination in milk. In this study, the nanoliposomes encapsulating Salvia oil (SO) has been prepared. Based on the damage effect of pore-forming toxin on cell membrane, α-toxin secreted from S. aureus has been used to trigger the release of SO from nanoliposomes to achieve antibacterial effect on S. aureus biofilm on milk container. Firstly, the minimum biofilm eradication concentration (MBEC) and biofilms time-dependent killing of SO were tested. The results showed that the MBEC of SO against S. aureus biofilms was 0.2% and SO effectively eradicated the biofilms after treating for 4 h. Subsequently, SO was encapsulated into nanoliposomes in order to increase its stability. The particle size, poly dispersity index (PDI), zeta potential, pH, turbidity, and entrapment efficiency of SO nanoliposomes were analyzed systematically. Gas chromatography–mass spectrometry (GC–MS) has been utilized to observe the controlled release of SO form nanoliposomes incubated with S. aureus. The scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) images have also visually showed that SO nanoliposomes have high anti-biofilm activity against S. aureus biofilms on milk container.     

Prevalence and characterization of Listeria monocytogenes isolated from retail food in Henan,China

Listeria monocytogenes, an important foodborne pathogen, is the causal agent of listeriosis. In this study, a total of 954 food samples originating from raw meat, cooked meat products, seafood, and vegetables purchased from supermarkets and open-air markets in Henan province, China, were analyzed for the presence of L. monocytogenes. All L. monocytogenes isolates were subjected to serotyping, pulsed-field gel electrophoresis (PFGE), and antimicrobial resistance. The overall percentage of L. monocytogenes prevalence was 6.2% (n = 59) with the highest rate of 7.4% for cooked meat products followed by raw meat (6.7%). The isolates belonged to five serotypes (1/2a, 1/2b, 1/2c, 4b, and 4c), with serotype 1/2a being predominant (55.9%). PFGE revealed a low genetic diversity among the isolates, irrespective of their sources, suggesting that dominant clones are widespread in different food products in Henan. Resistance to cefotaxime (30.5%) and ciprofloxacin (13.5%) was most often, whereas resistance to tetracycline, trimethoprim/sulfamethoxazole, and erythromycin was observed less frequently. The presence of L. monocytogenes in food products and antimicrobial resistance among the isolates represents a potential public health risk. Our results indicate that effective hygienic measures and bacteriological controls are necessary in China to reduce the contamination of retail food samples by L. monocytogenes.     

Development of an SD-PMA-mPCR assay with internal amplification control for rapid and sensitive detection of viable Salmonella spp., Shigella spp. and Staphylococcus aureus in food products

In this work, a specific, sensitive, and accurate technique was presented for simultaneous detection of Salmonella spp., Shigella spp., and Staphylococcus aureus in food products, three of the more frequent foodborne pathogens that were usually reported in a variety of food matrices. An internal amplification control (IAC) was added in a multiplex PCR (mPCR) reaction system as an indicator of false negative result that can come from the presence of PCR inhibitors in food products. In the presence of inhibitor, no signal would result for the target genes as well as the IAC which results in a positive signal, thereby, eliminating false negative results. To ensure detection of only the viable cells, the effects of sodium deoxycholate (SD) in combination with propidium monoazide (PMA) treatment in the presence of dead cells and viable cells were investigated. Results showed that PMA treatment alone could not effectively inhibit the detection of 10⁷ CFU/mL of dead Salmonella Typhimurium, Shigella sonnei, and S. aureus from PCR amplification. However, the SD in combination with PMA treatment gave negative results for PCR amplification of dead S. Typhimurium, S. sonnei, and S. aureus in pure culture and food products. When the developed SD-PMA-mPCR assay in combination with IAC was applied to detect the spiked food (milk, ground beef), the LOD of SD-PMA-mPCR assay for S. Typhimurium, S. sonnei, and S. aureus inoculated individually or inoculated simultaneously into milk or ground beef were 10¹ CFU/mL or 10¹ CFU/g after 15 h enrichment. The results suggested that the SD-PMA-mPCR assay in combination with IAC held promise for the detection of foodborne S. Typhimurium, S. sonnei, and S. aureus.     

Prevalence of Listeria spp. and antibiotic susceptibility of Listeria monocytogenes isolated from raw chicken and ready-to-eat chicken products in Jordan

Listeria monocytogenes is the causal agent of listeriosis, a disease that can be serious and is often fatal in susceptible individuals. The objective of the study was to determine the prevalence of Listeria spp. in raw chicken and ready-to-eat (RTE) chicken products in Amman, Jordan and the antimicrobial resistance of L. monocytogenes isolates. A total of 280 raw chicken and RTE chicken products (chicken-shawirma, chicken-burger, chicken-sausage and mortadella) were collected from Amman abattoir and local retail markets in Amman city. Listeria spp. were isolated by the conventional International Organization for Standardization (ISO) method and L. monocytogenes identified by biochemical and Polymerase Chain Reaction (PCR). Results of conventional method showed that out of total 280 samples, 141 (50%) were found to be contaminated with Listeria spp. [L. monocytogenes (18.2%), Listeria ivanovi (26.1%), Listeria grayi (3.5%), Listeria seeligeri (1.8), Listeria welshimeri (0.7%)]. The PCR confirmed all L. monocytogenes isolates (51 isolates: 15 from raw dressed broiler chicken, 23 from chicken-burger, 9 from chicken-sausage, and 4 from chicken-shawirma). Five of the tested L. monocytogenes isolates were resistance to two antibiotics (tilimicosin and tetracycline) among the ten tested antibiotics as determined by microbroth dilution method. The results presented in this study indicate the potential risk of contamination of RTE chicken products with L. monocytogenes.     

Development of a rapid and sensitive quantum dot-based immunochromatographic strip by double labeling PCR products for detection of Staphylococcus aureus in food

Staphylococcus aureus was listed as the 2nd common food-borne pathogens, resulting in diseases such as pneumonia, pseudomembranous colitis, pericarditis and even sepsis. A rapid, simple and sensitive detection method is required to monitor food in cases of contamination by S. aureus. Hence, a novel immunochromatographic test strip (ICTS), based on the two-component reaction (i.e. digoxigenin and anti-digoxigenin, quantum dots-conjugated streptavidin and biotin) in test line with QDs as indicator, was developed for the rapid and sensitive detection of S. aureus. Genomic DNA from bacteria lysis by boiling was extracted easily and rapidly with silica-coated magnetic nanoparticles, and a species-specific gene was amplified by PCR using digoxigenin/biotin-labeled primers. The fluorescence of captured QD labels on the test line and control line served as signals was observed by UV light. The sensitivity and specificity of the ICTS were estimated using bacteria spiked food samples (artificially mixed with cell counts (10⁸ CFU/mL for each bacterium) of Escherichia coli, Listeria monocytogenes and Lactobacillus plantarum). The limit of detection for S. aureus was 3 × 10⁰ CFU/mL and 3 × 10¹ CFU/g in spiked milk powder and meat samples, respectively, which was not affected by those non-S. aureus strains. Our results showed that QDs-based ICTS was promising for rapid and sensitive detection of S. aureus within 2 h. Hence, this protocol might be useful for screening and monitoring the contamination of S. aureus in food products, and helpful for promoting the prevention and control of communicable disease caused by food-borne pathogen.     

The prevalence and serovar diversity of Salmonella in various food products in Estonia

In this work the prevalence and serovar diversity of Salmonella in various food products including non-thermally processed food and ready-to-eat (RTE) food in Estonia in 2008–2012 are summarized. The findings demonstrate that the overall prevalence of Salmonella in these food categories was low. A total of 260 (0.54%) of 47,927 food samples were found to be positive for Salmonella, the overall prevalence in non-thermally processed food was 0.81% (256/31,576) and in RTE products only 0.02% (4/16,351). Salmonella was most often isolated from raw eggs and products thereof (2.17%, 5/230), followed by raw meat products (0.95%, 207/21,723), RTE mayonnaises (0.90%, 2/221) and raw meat (0.89%, 38/4252). In the raw meat category, Salmonella was most frequently isolated from turkey meat (6.96%, 11 positive samples out of 158), broiler chicken meat (4.00%, 7/175) and from layer hen meat (2.22%, 11/496). Salmonella was isolated in lesser extent from meat preparations (1.91%, 82/4292), minced meat and mechanically separated meat products (0.97%, 100/10,344) and from raw sausages (0.35%, 25/7087).Altogether 24 different serovars were identified among the 260 Salmonella positive samples. Salmonella Typhimurium was the most frequent serovar (26.90% of the positive samples) and it was isolated most commonly in raw food products. The next most frequent serovars were Salmonella Derby (17.50%), Salmonella Enteritidis (8.37%) and Salmonella Newport (7.57%). The only serovars isolated from the Salmonella positive RTE food samples were Salmonella Infantis (two isolates) and S. Enteritidis (two isolates).