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Comparative analysis of Staphylococcus aureus strains by molecular microbiology methods
34-39Views:108Staphylococcus aureus is a very important pathogen for dairy farms and milk processing plants. Subclinical mastitis is often caused by this species, and it can contaminate bulk tank milk when milking cows are suffering from mastitis. Additionally, thermostable enterotoxins (SE) produced by some types of this bacterium can cause food poisoning.
The aim of our research was to examine the number of S. aureus in bulk tank milk in two dairy farms and the enterotoxin-producing ability, genetic relation (pulsotype) and antibiotic resistance of S. aureus strains from different sources (bulk tank milk, udder quarter milk and environment).
The results show that the mean number of S. aureus of bulk tank milk of two farms significantly differed (P<0.05). Fourteen isolates were selected for further molecular genetic studies (five isolates were from bulk tank milk and nine isolates were from udder quarter milk). S. aureus was not recovered from the environmental samples. Three of the fourteen isolates (21.4%) tested by multiplex PCR were positive for SE genes. Two isolates carried one gene (seb) and one isolate carried two genes (seg and sei). The fourteen strains were classified into three pulsotypes and two subtypes at 86% similarity level. Isolates from bulk tank milk (n=5), were divided into 2 pulsotypes (A, C) and one subtype (C1). The isolates from udder quarter milk (n=9) belonged to three different pulsotypes (A, B, C) and two subtypes (A1, C1). The distribution of pulsotypes in the present study revealed genetic relationship between S. aureus isolated from udder quarter milk and bulk tank milk. This could be explained by the fact that in farms with a high number of infected cows, these cows could represent the main source of contamination. The results of the antibiotic resistance investigations show, that all strains were susceptible to methicillin, cefoxitin, lincomycin, tetracycline, erythromycin and sulfamethoxazole/trimethoprim. Thirteen out of fourteen strains were resistant to penicillin (A and C pulsotypes, A1 and C1 subtypes) and just one isolate was susceptible (B pulsotype) to all antibiotics tested. -
Comparison of sample preparation methods for the identification of Staphylococcus Aureus by MALDI-FOF MS
9-14Views:267Coagulase-positive staphylococci include 3 species, Staphylococcus aureus, S. hyicus and S. intermedius. Of these three species, S. aureus is the most well-known human pathogen. S. aureus is part of the human and animal normal microbiota, however, it is capable of producing several staphylococcal enterotoxins (SEs) that cause intoxication symptoms of varying intensity in humans after consuming contaminated food. Selective media which are used for the determination of coagulase-positive staphylococci from foods are not able to identify isolates at a species. With the MALDI-TOF MS technique, we can identify S. aureus cheaper and faster than by using molecular methods. This paper describes the results of the study of the presence of coagulase-positive staphylococci and S. aureus in many food products, and the application of three sample preparation methods: direct sample preparation, formic acid suspension and ethanol extraction.
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The effect of season on the microbiological status of raw milk
95-99Views:117Many factors can influence the microbiological quality of raw cow’s milk. In this study, our aim was to determine whether there was any difference between the microbiological statuses of milk produced in different seasons. Samples were collected and analysed from five dairy farms in Hajdú-Bihar County, from February to November in 2019. During our studies, total plate count (TPC), coliform count and Staphylococcus aureus count of raw cow’s milk samples were determined.
There was no significant difference (P>0.05) between the mean TPC values detected in the milk collected in winter and autumn, but that values were significantly (P<0.05) lower than in the milk samples collected in spring and summer. Similarly to the TPC, in the case of coliform bacteria the lowest mean colony count was detected in the samples collected in winter. The difference was significant (P<0.05), compared to the values observed in the samples collected in summer. S. aureus was detected in bulk milk of only two farms in excess of 1.0 log10 cfu/ml. Also in case of S. aureus, there was a significant difference (P<0.05) between the values observed in the samples collected in winter and in summer. Samples from spring and summer contained the highest amount of S. aureus.
Based on the results of our studies, in the case of almost all farms the mean TPC, coliform and S. aureus counts were lower in the samples collected in winter, than in the samples collected in summer. The fact that the samples collected in winter contained the lowest amount of colonies could be attributed to the inhibition of growth of mesophilic microorganisms below 8 °C. Furthermore, the fact that we observed the highest colony counts in samples collected in summer, can be related to the heat stress of cows during the summer due to unfavorable weather conditions (high temperature and humidity).
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The effect of breed and stage of lactation on the microbiological status of raw milk
37-45Views:296The microbiological quality of the milk is important not only for food safety, but it can also influence the quality of dairy products. The microbiological status of raw cow milk can be influenced by many factors. Our aim was to determine whether there was a difference between the microbiological quality of milk of two different cow breeds (Holstein Friesian and Jersey) kept and milked in the same conditions, and how the microbiological quality of the raw cow milk changed during lactation (beginning, mid, and end). Samples were taken and analysed in July, August and September in 2018 from two dairy farms in Hajdú-Bihar county. During the conducted studies, the total plate count (TPC), the coliform count, the Staphylococcus aureus count and the coagulase-negative Staphylococcus (CNS) count of raw milk samples were determined.
There was no significant difference (P>0.05) between the milk of the Holstein Friesian and Jersey breeds in the case of TPC. However, the mean coliform count of milk samples taken from Holstein Friesian cows was significantly lower (P<0.05) than the mean coliform count of milk samples taken from Jersey cows. S. aureus was detected in one of the twelve milk samples taken from Holstein Friesian cows, and in two of the eleven milk samples taken from Jersey cows. CNS was found in larger amount in milk samples taken from Holstein Friesian cows, and the difference was significant (P<0.05). Both TPC and CNS count were significantly higher (P<0.05) in individual milk samples taken at the end stage of lactation, than in samples taken in the earlier stages of lactation from Farm “A”. However, in the case of Farm “B”, there was no significant difference (P>0.05) in colony counts at different stages of lactation. S. aureus was only present in milk samples that collected from cows, which were at the beginning and middle stages of lactation. Testimg the hemolysin production ability of S. aureus strains isolated from the raw milk samples, only weak hemolysis was observed on blood agar. In case of antibiotic resistance testing, it was found that all strains were susceptible to cefoxitin, chloramphenicol, clindamycin, erythromycin, gentamicin, penicillin G, tetracycline and trimethoprim/sulphamethoxazole.
Based on the results of our studies, staphylococci were detected in a higher amount in the milk of Holstein Friesian cows, and coliform bacteria were detected in a higher number in the milk of Jersey cows. Summing up the results of the milk samples taken from the different stages of lactation in one of the farms, it can be concluded that higher TPC and CNS count could be detected at the end stage of lactation than in the samples taken from the earlier stages of lactation. The fact that at the end of lactation the microorganisms could be detected in a higher colony count may be related to the fact that teats could be damaged during lactation by the milking machine, which increased the chance of imvading the microorganisms into the udder.
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Microbiological status of bulk tank milk and different flavored gomolya cheeses produced by a milk producing and processing plant
73-78Views:228The microbiological quality of milk is important not only for food safety, but it can also influence the quality of dairy products. In this study, our aim was to assess the microbiological status of the bulk milk of a milk-producing farm, and some natural and flavored (garlic, dill, onion) gomolya cheeses made from pasteurized milk produced by their own processing plant. We determined the number of coliform bacteria, Escherichia coli, Staphylococcus aureus, and molds of three milk and eight cheese samples. The tests were conducted between July and September, 2017.
In bulk milk, the mean coliform count was 3.83±0.17 log10 CFU/ml; the mean E. coli count was 1.38±0.14 log10 CFU/ml; the mean mold count was 3.74±1.30 log10 CFU/ml; and the S. aureus count was <1.00 log10 CFU/ml, respectively. The mean coliform count in gomolya cheeses was 3.69±1.00 log10 CFU/g; the mean E. coli count was 2.63±0.58 log10 CFU/g; the mean S. aureus count was 3.69±1.35 log10 CFU/g and the mean mold count was 1.74±0.37 log10 CFU/g. The amount of coliforms detected in different flavored gomolya cheeses were significantly different (P<0.05). More than 10 CFU/g of E. coli was found only in the dill flavored cheeses, and S. aureus was found only in dill (3.66±1.86 log10 CFU/g) and onion (3.71±0.52 log10 CFU/g) flavored gomolya cheeses. Based on the obtained results, it was found that the amount of coliform bacteria and E. coli in bulk milk exceeded the limit set in regulation of the Hungarian Ministry of Health (MoH) 4/1998 (XI. 11.) and the amount of S. aureus was below the limit. For gomolya cheeses, the S. aureus count exceeded the limit. The amount of coliform bacteria remained above the limit in cheeses, except for the garlic flavored gomolya cheese. In cheeses, a larger E. coli count was detected than in the bulk milk, but there is no specific limit for cheeses in the regulation. The mold count exceeded the limit specified in the regulation in cheeses, but a lower value was detected relative to milk.
The results show that, in the case of bulk milk and gomolya cheeses, certain detected quantities exceeded the limit values set forth in regulation of MoH 4/1998 (XI. 11.). The results indicate an inadequate microbiological state of the raw material and the finished products. The reasons for these are due to reduced technological hygiene or the inappropriate handling of raw material and finished products. In this study, we have summarized the results of our preliminary studies, which can provide a basis for further hygiene studies.
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Microbiological quality of bulk tank raw milk from two dairy farms in Hajdú-Bihar County, Hungary
105-112Views:289Two main channels have been identified to be responsible for microbiological contamination of raw milk and milk products. Firstly, contamination has occurred due to udder infection from the cow or the blood which harbours most bacteria that come in contact with the raw milk. Secondly, via external factors (may include faeces, skin, contaminated water, environment etc.) which are associated with the operation of milking. There is direct contact with the milk and/or surfaces before, during or after the milking, posing public health risk and economic decline. The aim of this study was to examine the bacteriological quality of bulk tank raw milk samples collected from two different size dairy farms (Farm 1 and Farm 2) of different housing forms (cubicle loose and deep litter) in Hajdú-Bihar County, Hungary in July, 2017. Three samples were taken from each farm, and the total plate count, coliform count, Escherichia coli count, Staphylococcus aureus count, and yeast and mould count were determined in them.
The results clearly showed low level of all measured bacteria group load in Farm 1 samples in comparison to Farm 2 with the exception of coagulase-negative Staphylococcus (CNS) which represented high level in general, indicating significant difference (P<0.05). The mean value of total plate count in Farm 2 samples was higher (1.0 × 105 CFU/mL) than Farm 1 samples (2.8 × 104 CFU/mL). There was a significant difference (P<0.05) in mean count of coliforms in raw milk samples between Farm 1 and Farm 2. Similarly, results of E. coli were significantly different (P<0.05) with mean count of 1.44 × 102 CFU/mL and 2.02 × 103 CFU/mL for Farm 1 and Farm 2 respectively.
Results of Staphylococcus aureus also showed significant difference (P<0.05) with mean count of 9.7 × 101 CFU/mL for Farm 1 and 6.28 × 102 CFU/mL for Farm 2. The mean of mould count recorded was 1.07 × 102 CFU/mL and 4.93 × 102 CFU/mL for Farm 1 and Farm 2 respectively. The recorded mean of yeast count was 1.68 × 103 CFU/mL and 3.41 × 103 CFU/mL for Farm 1 and Farm 2 respectively; however, both farms showed no significant difference (P>0.05) in terms of mean of mould and yeast count. Although Farm 2 produced six times lower milk quantity than Farm 1, the measured microbial parameters were high. Both farms’ microbiological numbers were higher above the permitted limit values as stated by Regulation (EC) No 853/2004, Hungarian Ministry of Health (MoH) 4/1998 (XI. 11.).
This could be an indication of non-conformance to effective GMP, ineffective pre–milking disinfection or udder preparation, poor handling and storage practice, time and temperature abuse and inadequate Food Safety Management System Implementation. Therefore, our recommendation is as follows; establish control measures for pre- and postharvest activities involved in the milking process which would be an effective approach to reduce contamination of the raw milk by pathogenic microorganisms from these farms, strict sanitation regime and hygiene protocol be employed and applied to cows, all equipment, contact surfaces and minimize handling of the milk prior, during and after milking. This will also serve as scientific information to the producers for continual improvement in their operations.
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Effect of plant extract against opportunist human pathogen soil bacteria
89-93Views:99Our experiments have repeatedly shown that the extract of ragweed (Ambrosia artemisiifolia) is an antifungal and antibacterial component.
In our paper we tested of the biological activity of the herbal substance against opportunistic human pathogenic bacteria strains (Staphylococcus
aureus 110003 and 25923; Staphylococcus saprophyticus 110008). Our laboratory tests show that the extract is bacteriostatic and in several cases bactericid. We can assume that from the the agricultural and public health aspects can be extremely dangerous weed, contains biologically active components and it may be suitable for the prepare of antibacterial agents. -
Testing the antimicrobial activity of essential oils
71-74Views:129The vapor phase of some essential oils proved to have antimicrobial activity. Utilization of the vapor phase of Eos is presently understood as one of the possible alternatives to synthetic food preservatives which could be used in the future. However, testing the vapor phase of EOs against microorganisms causing food-borne diseases (e.g. Salmonella enteritidis or Staphylococcus aureus) or food spoilage is relatively new. Consequently, due to the large number of known EOs, research on their antimicrobial activity is still largely in the phase of in vitro rather than in vivo testing. Moreover, no standard and reliable method for fast screening of a wide range of samples exists. Thus, the aim of this study is to show results concerning tests of the antimicrobial activity of EOs against S. enteritidis or S. aureus, which were conducted by two modifications of the disc volatilization method we developed. The lately developed method has the potential to become widely used for fast screening of EO antimicrobial activity in the vapor phase.
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Antimicrobial effect of dried sage on the microbiological state of fresh Hungarian sausage
189-192Views:109The purpose of this study was to evaluate the microbial effect of dried sage (Salvia officinalis L.) on the traditional Hungarian sausage. We added 0.5, 1, 1.5, and 2 w/w% of sage to the sausages and tested them on the 0th, 7th and 14th day. The added dried sage had no effect on the tested microorganisms, but the sage extract inhibited Salmonella, Enterococcus faecium and Staphylococcus aureus.
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Bacteria in the milk of sheep with or without mastitis- mini Review
47-52Views:302From a nutritional point of view, sheep milk is more valuable than cow and goat milk and the interest for sheep milk is increasing in many countries. However, sheep milk is easily contaminated during milking, handling, and transport and it is an ideal medium for bacterial propagation. Consequently, sheep milk spoils quite quickly. The proper, clean handling of milk is not only of sanitarian interest, but it also serves the farmers’ interests, because contaminated milk may not be distributed, and is unsuitable for producing good quality products. Following this technological trend, this review addresses the bacterial composition of sheep milk with and without mastitis. Even though sheep milk contains a lot of bacteria, this review article highlighted total plate count, Enterobacteriaceae, coliform, Staphylococcus aureus, Escherichia coli, Listeria monocytogenes, Campylobacter, Salmonella spp. and Streptococcus spp. Mastitis in sheep is a vital cause of mortality, reduction in milk production and early culling. The reported risk factors for mastitis in sheep were age, a case of mastitis, breed, husbandry systems, and location. The main priority should be implementation of programs to minimize human pathogenic bacteria and mastitis in raw ewe milk.
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The effect of keeping technology on the microbiological status of raw milk
67-75Views:100The importance of the quality of raw milk increased after Hungary had joined to the EU. On delivery of raw milk, the microbiological quality, especially total plate count of the milk is very important. Twenty-two farms (7 large, 4 medium-sized, and 11 small farms) were included in the study. We considered the different farm size, keeping- and milking circumstances during the selection of farms. The examined large farms use loose housing system (cubicle, deep litter) and milking parlour. Most of them use preand post-milking disinfection. In the medium-sized farms, loose,
deep litter and tie-stall housing system, as well as milking parlour, pipeline milking and bucket milking occurred. All of them use preand post-milking disinfection. Small farms use tie-stall housing system, bucket milking and udder preparation by water. Unfortunately, they do not use pre- or post-milking disinfection. In the large and medium-sized farms mainly Holstein Friesian, in the small farms Hungarian Simmental breeds can be found.
The aim of our research was to examine the microbiological status of the raw milk produced in dairy farms (total plate count, coliform count, Escherichia coli count, Staphylococcus aureus count, psychrotroph bacteria count, furthermore yeast and mold count); sources of the contamination; connection between the microbiological quality of produced milk and housing-, milking technologies of farms; furthermore the hygienic circumstances of milking and milk handling of the farms, by the examination of coliform bacteria and Escherichia coli contamination.
During the examination of the connection between the different farm sizes, various housing- and milking forms and the microbiological characteristics we observed similar tendencies in the case of total plate count, coliform count, yeast and molds count, furthermore psychrotroph bacteria count. The value of these parameters was significantly higher in small farms, and infarms which use tie-stall housing forms, bucket milking, udder preparation with water, and which do not use pre- and post-milking disinfection.
The results showed that besides cooling, the milking procedure and the type of udder preparation had the largest effect on the total plate count. Statistical analysis shows that in medium and small farms the combination of pipeline milking – tie stall housing system – disinfectant preparation of the udder; in large farms the combination of milking parlour – loose cubicle housing system – dry preparation of the udder are the most appropriate in the aspect of the total plate count. We experienced that in farms where the hygienic instructions are not followed – and therefore
equipment used during the milking and handling of milk is very contaminated – or rather the separation of mastitic cows’ milk is not appropriate, different microorganisms may contaminate the produced milk. -
Definition antioxidant activity of selenium-enriched food sprout, as well as their microbiological analysis
25-30Views:179In this present study, we prepared selenium-enriched food sprouts, where the antioxidant capacity was analysed, we also determined their microbiological status. We took into account the fact, we choose micronutrients to our treatment, that selenium can be delivered to the body by a small amount with the most widely consumed food.
We focused during our research to determine that the increasing concentrations of selenium treatment, in which we used sprouts, knowing fully well that it has an impact on aboriginal antioxidant capacity of sprouts, which is mainly due to high vitamin content of sprouts.
Furthermore, we think it is important to make microbiological analysis, because germination conditions, for example temperature, pH, all this will create an ideal environment for the growth of microorganisms. So we had goal to determine, how the used selenium concentration affect the total plate count, coliform bacteria count and Staphylococcus aureus count of sprouts.
We determined the aboriginal water-soluble and lipid-soluble antioxidant capacity of sprout with the PHOTOCHEM chemiluminometer and we applied pour plate technique for the mapping of the mycrobiological state of sprouts.
Experimental results are evaluated, we state that increasing concentrations of selenite or selenate treatment, is primarily water-soluble antioxidant capacity of sprouts was affected. The water-soluble antioxidant capacity of wheat sprout was much higher than the measured values in pea sprout, this may be linked to what we measured. That is much higher ascorbic acid content in case of wheat sprout, which is well known as one of the most important antioxidant properties compounds of wheat sprout.
We conclude from the results of the microbiological, that the highest concentrations of selenite or selenate treatment has a relative significant anti-microbial effect in case of wheat sprouts. Coliform and total plate count showed no clear decreasing tendency, although the values of treatments in both cases obtained were below the control values.