Baird-Parker Agar Research Articles

Species composition and methicillin resistance of staphylococci taken on dairy farms

Methicillin-resistant staphylococci can often asymptomatically colonize animals and humans and are capable of causing disease in them. Therefore, their identification and species identification are important for establishing the source of zoonotic infection and the reservoirs of antimicrobial resistance genes. The purpose of the search was to study the spread of methicillin-resistant staphylococci on dairy farms in the Western region of Ukraine. BD Baird-Parker Agar (HiMedia, India) was used to isolate staphylococci. Specific identification of pure cultures was performed using “RapID Staph Plus” kits (Oxord, UK). Staphylococcus sensitivity to methicillin was determined by inoculum application on Muller-Hinton agar with oxacillin (HiMedia, India). The sensitivity of the isolates to antibacterial preparations was determined by disco-diffusion method. The results of our searches show that Staphylococcus aureus is virtually identical in the amount both from cows (50.1 %) and from humans (62.4 %). In this case the frequency of its isolation among other species was 20.3 %. Along with Staphylococcus aureus there are such species as: S. haemolyticus (20.3 %), S. saprophyticus (13.6 %), S. xylosus (14.0 %), S. chromogenes (11.1 %), S. sciuri (8.8 %), S. epidermidis (4.8 %), S. hominis (3.4 %), S. cohnii (2.6 %) and S. warner (0.7 %). In this case, approximately the same irradiation of cows, humans and the environment by species S. haemolyticus (44.5:70.8:58.8 %), S. epidermidis (12.7:16.6:9.1 %), S. xylosus (26.0:37.4:52.9 %) is observed. The share of S. aureus strains on methicillin-resistant dairy farms in the Western Ukraine is 26.8 %. The proportion of S. aureus strains on methicillin-resistant dairy farms in the Western Ukraine is 26.8 %. Methicillin resistance is also shown S. haemolyticus, S. saprophyticus, S. xylosus та S. chromogenes. In this case their number is 1.1, 1.3, 1.6 and 5.5 times lower, respectively, and S. hominis 1.2 times higher than S. aureus. In addition, the selected cultures simultaneously show resistance to two or more antibiotics. Thus, staphylococci circulating on dairy farms are a large reservoir of resistance genes of antimicrobial preparations. Therefore, it is necessary to establish a constant control of the secretion of staphylococci resistant to β-lactam antibiotics.

Isolation and Molecular Identification of Staphylococcus Species in Cow’s Milk Distributed in Khartoum State

The aim of this study was to identify and evaluate the bacteria load and the level of Staphylococcus Species and Staphylococcus aureus contamination in raw cow´s milk distributed in Khartoum state. One hundred and eighty samples were taken from Khartoum, Omdurman and Khartoum North, 60 samples of raw milk from healthy apparently cows in farms, 60 samples of raw milk vended by donkeys, 60 samples of raw milk vended by cars. Total Viable Bacterial count was done using standard plate count. The samples were detected for the presence of Staphylococcus Species using conventional methods, cultured in Baird-Parker and Mannitol agar, Gram Stain, biochemical tests were done. The TVBC showed that the highest bacterial load was detected in the raw milk vended by Donkey (6.90±.03 log10 cfu/ml) vended by cars (6.78±0.12 log10 cfu/ml) then the lowest bacterial load detected in the milk collected from farm (6.63±0.07 log10 cfu/ml). Out of 180 samples of raw milk studied, 130 showed contamination by Staphylococcus species corresponding to 72.2% of the samples being contaminated and out of 180 samples of raw cow´s milk 80 was contaminated with staphylococcus aureus corresponding to 44.4% of the samples.The isolated Staphylococcus species was confirmed further by using the Polymerase Chain Reaction (PCR) targeting the partial sequence of 16s rRNA gene. Sequencing identified Staphylococcus aureus, staphylococcus epidermidis, Staphylococcus hominies and Staphylococcus simulans. The results showed a high level of contamination by Staphylococcus Spp. and staphylococcus aureus in raw cow´s milk that distributed in Khartoum state.

Исследование вагинального микробиома женщин с привычным невынашиванием беременности, а также спектра выделяемых ими газовых сигнальных молекул

Введение. Проблема невынашивания беременности в настоящее время не теряет своей актуальности. Частота данной патологии по оценкам различных авторов составляет от 2 до 5% в общей популяции. Как известно, инфекционный фактор играет весьма значимую роль в генезе привычного невынашивания. Вопрос об этиологической роли инфекции в привычном невынашивании широко дискутируется в литературе. Цель исследования - анализ микробиома влагалища здоровых женщин и женщин с привычным невынашиванием беременности, а также спектра выделяемых лактобациллами газовых сигнальных молекул, играющих важную роль в поддержании здоровой жизнедеятельности организма. Методика. Забор материала из влагалища производили стерильным тампоном на полистироловой палочке с площади 1 см2 и в течение 2 ч доставляли в бактериологическую лабораторию. Для выделения факультативно анаэробных и аэробных бактерий использовали питательные среды: Эндо агар для энтеробактерий, маннит-солевой агар (М118) для стафилококков, микрококков, для выявления лецитиназной активности - агар Бэрда-Паркера и так далее. Культивирование проводили при температуре 37 °С в течение 24-48 ч. Количество колоний выражали в lg КОЕ/мл. Продукцию газовых сигнальных молекул (H2, O2, N2, CO, CH4, CO2, NO, H2S) определяли с помощью метода газовой хроматографии на приборе Хроматэк-кристалл 5000.2. Количество выделенных газов измеряли в ppm (млн-1) . Результаты. Спектр основного микробиома влагалища здоровых женщин в возрасте 19-23 лет представлен бактериями нормальной микробиоты родов Lactobacillus, Enterococcus, Bifidobacterium, Peptococcus, Peptostreptococcus, Bacillus, Staphylococcus (эпидермальные штаммы). Условно-патогенные грибы рода Candida, бактероиды, золотистый стафилококк, стрептококки, микрококки, вейлонеллы, гарднереллы и актиномицеты выделялись в редких случаях. Различные штаммы лактобацилл выделялась у 91% здоровых женщин. Среди газовых молекул, продуцируемых лактобациллами, преобладали: СО2, СО и NO. Установлены дисбиотические нарушения микробиома влагалища у небеременных женщин с привычным невынашиванием беременности в анамнезе. Также у данной группы пациенток отмечено снижение продукции газовых сигнальных молекул: СО и NO, и повышение продукции H2S и CH4. Выводы. Полученные результаты свидетельствуют об угнетении иммунного ответа у данной категории больных и необходимости коррекции дисбиотических нарушений еще на этапе прегравидарной подготовки.Background. The issue of habitual miscarriage is very important at the present time. Prevalence of this pathology is approximately 2-5%. Infection considerably contributes to the pathogenesis of habitual miscarriage. The etiology of infection has been widely discussed in the literature. Aim. To study the vaginal microbiome of healthy women and women with habitual miscarriage as well as the spectrum of gaseous signal molecules released by lactobacillus, which is essential for maintaining healthy vital activity. Method. Samples of vaginal material were collected with a sterile swab on a polystyrene stick from an area of 1 cm2; the samples were delivered to the bacteriological laboratory within 2 hours. Facultative anaerobic and aerobic bacteria were isolated using the following nutrient media: Endo agar for enterobacteria; mannitol-salt agar (M118) for staphylococci and micrococci; Baird-Parker agar for detection of lecithinase activity, etc. Cultivation was performed at 37°C for 24-48 hours. The number of colonies was expressed in l g CFU/ml. Production of gaseous signaling molecules (H2, O2, N2, CO, CH4, CO2, NO, and H2S) was determined using a Chromatech Crystal 5000.2 gas chromatograph. The amount of gases was measured in ppm. Results. The major vaginal microbiome spectrum of healthy women (19-23 years) was represented by normal microbiota bacteria of the genera Lactobacillus, Enterococcus, Bifidobacterium, Peptococcus, Peptostreptococcus, Bacillus, and Staphylococcus (epidermal strains). Opportunistic fungi of the genera Candida, Bacteroides, Staphylococcus aureus, Streptococci, Micrococci, Weylonella, Gardnerella, and Actinomycetes were found rarely. Lactobacillus strains were isolated from 91% of healthy women. Lactobacilli produced most of CO2, CO, and NO molecules. Dysbiotic vaginal microbiome was found in non-pregnant women with a history of habitual miscarriage. These patients also had decreased production of CO and NO, and increased production of H2S and CH4. Conclusion. The study demonstrated suppression of the immune response in this category of patients. Dysbiosis requires correction prior to pregnancy.

Antimicrobial effect of the essential oil of Oregano against Listeria monocytogenes and Staphylococcus aureus.

The food industry uses chemical compounds to prevent the growth of bacteria and extend their shelf life. These substances are associated with toxic problems, the presence of chemical residues and antimicrobial resistance. Oregano essential oil has antimicrobial compounds such as thymol and carvacrol. The objective of the research was to evaluate the antimicrobial effect of oregano essential oil from the province of Concepción, Junín Region (Peru) against Listeria monocytogenes and Staphylococcus aureus. Oregano was taxonomically identified as O. vulgare. The essential oil was obtained by hydrodistillation and the profile of chemical compounds was determined by Gas Chromatography/Mass Spectrometry (GC-MS). The minimum inhibitory concentration (MIC) was determined by microdilution and the minimum bactericidal concentration (CMB) by agar planting Baird-Parker Agar was used for S. aureus and for L. monocytogenes the DNA hybridization method was used using the Gene Quencekit® L. monocytogenes. The GC-MS profile of oregano oil had 11.9% thymol and 1.7% carvacrol. The oil exhibited antimicrobial activity against S. aureus (CMI 2%, CMB 4%) and L. monocytogenes (CMI-4%, CMB-4%). The high percentage of CMI and CMB was due to the low concentration of carvacrol and thymol, however this oil can be used in the food industry as a natural antimicrobial and/or disinfectant.

Foodborne Methicillin-Resistant Staphylococcus Aureus (FB-MRSA) in Skimmed Dairy Products in Egypt: Prevalence, Molecular Characterization, and Antimicrobial Susceptibility

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the emerging pathogens worldwide. The aim of this study was to investigate the prevalence of MRSA among different skimmed dairy products retailed in local markets in Alexandria City, Egypt. Two hundred and ten samples including Kareish cheese, light Feta cheese, light processed cheese, light yoghurt and skimmed milk powder were examined for this purpose by surface spread technique on Baird-Parker agar plates. Further molecular detection of determinant genes of S. aureus (23S rRNA), MRSA (mecA), enterotoxigenic genes B and D (seb, sed) and antimicrobial susceptibility patterns were carried out. Out of 210 examined samples, MRSA could detect in 7 samples, meanwhile enterotoxigenic genes could not detect in all isolated strains. All MRSA isolates were phenotypically resistant to Oxacillin. Meanwhile, 85.7% were susceptible to Trimethoprim/Sulfamethoxazole. Multidrug-resistant was reported in all isolated MRSA strains in this study which may constitute a public health concern. Therefore, strict hygienic measures should be adopted and carried out to preserve such skimmed dairy products from emerging multidrug-resistant MRSA and to ensure food safety.

MICROBIOLOGICAL QUALITY OF GOAT MILK PRODUCED ON ALAGOAS STATE, BRAZIL

The objective of this study was to verify the microbiological quality of raw and pasteurized goat milk and produced in Alagoas state, Brazil. Was carried out three interval collect of pasteurized and raw milk and the samples taken to the laboratory. All samples were subjected to aerobic mesophilic microorganisms counting, psychrotrophic microorganisms, determination of the most probable number (MPN) of total coliforms and thermotolerant microorganisms. For analysis, samples were plated on specific medium (Baird-Parker Agar Base himedia M043) and incubated at 37 °C for 24h. To Salmonella sp. detection was used plating the medium Salmonella Shigella agar. The presence of coliforms at 35 °C was detected in all samples as well as 45 °C except to first sample. To the pasteurized milk was found to coliforms at 35 °C and 45 °C in the second and third samples. Only in the second test was checked the presence of mesofilic bacteria. We have not found samples contaminated with microorganisms psychrotrophic, Salmonella sp. and Staphylococcus sp. According to the obtained results the quality of collected milk has unacceptable conditions of consumption compared to coliform counts, according to the parameters established by law.

Detection Staphylococcus aureus Producing Enterotoxin A on the Skewers Meatballs Product in Yogyakarta City Indonesia

Meatballs are snacks that are sold by street vendors in the school area and some public places in Yogyakarta city. Based on the materials used and the traditional producing process, it is possible that the hearty snacks can be contaminated by enterotoxin-producing Staphylococcus bacteria. This study aims to detect the presence of enterotoxin-producing Staphylococcus on skewers meatballs. Skewed meatball samples were taken from ten different locations in Yogyakarta city. The samples were grown on the Staphylococcus sp test standard medium, Baird Parker Agar (BPA). The colony candidates were then selected into Mannitol Salt Agar (MSA), carbohydrate fermentation medium, gram staining and then confirmed using the API Staph. Molecular characterization by detecting nuc genes and sea genes as encode enterotoxin A producers. The result of this research showed that the total bacteria ranged from 1.7x104 - 2.0x109 CFU/g. Identification results using API Staph were found as S. aureus, S. cohni, S. xylosus, S. lentus, S. warneri, and S. sciuri while nuc and sea genes were only found in S. aureus.

Assessment of the Holothuria atra (Echinodermata: Holothurians) habitat based on the antibacterial effectiveness approach at Karimunjawa, Central Java Province, Indonesia

Abstract. Sulardiono B, Anggoro S, Rudiyanti S, Rahman A. 2019. Assessment of the Holothuria atra (Echinodermata: Holothurians) habitat based on the antibacterial effectiveness approach at Karimunjawa, Central Java Province, Indonesia. Ocean Life 3: 11-17. Sea cucumber Holothuria atra lives in shallow marine benthic habitats and in its life cycle depends on the quality of its environment. Bacteria in the body of sea cucumbers come from the bioaccumulation of bacteria in the sediment that they exposed to through the process of absorption of sediments as a deposit feeder. To inhibit bacterial growth in the body, sea cucumbers release bioactive compounds as antibacterial. The purpose of the study is to determine the effectiveness of sea cucumber inhibition ability of sea cucumber H. atra as an antibacterial in the waters of Karimunjawa. A total of 15 individual sea cucumbers were taken from the waters of Menjangan Besar and 20 individuals from the Alang-alang waters, then cleaned and dissected to take the part of the body wall. The body wall samples obtained from each observation station was as much as 1 kg, which was then sliced into small pieces and preserved in 96% ethanol, and and the bioactive compound was extracted by maceration method. Antibacterial activity test was carried out by disc diffusion method on paper discs (10mm) and using the media of EMBA (Ethylene Methyl Blue Agar) (Merck) for E. coli bacteria and BPA (Baird Parker Agar) for S. aureus bacteria. The obtained antibacterial test results were based on the measurements of the formed clear zone diameter, so that it can be known the effectiveness status of the antibacterial sea cucumbers of each observation station. The results of the study show that the antibacterial effectiveness of sea cucumber H. atra in the Menjangan Besar waters is higher than in the Alang-alang waters. The increase of sea cucumber bioactive compounds in the waters of Menjangan Besar shows that the waters are suitable for the development zone of sea cucumber cultivation

Efficiency of PetrifilmTM Staph Express Count Plate for the Enumeration of Staphylococcus aureus in Meat, Fishery Product, and Korean Traditional Foods

To enumerate Staphylococcus aureus in food, Baird-Parker Agar (BPA) is usually used in the conventional method, However it requires time and space for the preparation and plating, and incubation. Thus, use of the 3MTM PetrifilmTM Staph Express Count Plate (STX Petrifilm) might be appropriate to solve these challenging problems. The purpose of this study was to compare the efficiency of STX Petrifilm with BPA for enumeration of S. aureus in various foods. A mixture of S. aureus strains ATCC29213, ATCC25923, and ATCC13565 was inoculated on marinated pork chop, beef (chuck tender), dried filefish, semi-dried squid, rice cake, and Japchae (stir-fried glass noodles) at 2, 3, 5, and 7 Log CFU/g. S. aureus cell counts were enumerated by spread-plating on STX Petrifilm and BPA after 0 and 24 hours at 4oC (marinated pork chop, beef, semi-dried squid, and stir-fried glass noodles) and 25oC (dried filefish and rice cake). Recovery of STX Petrifilm for S. aureus from various food samples was compared with BPA, and the results showed that there were no significant differences between two selective media in all cases. The results indicated that STX Petrifilm had enough efficiency to recover S. aureus from various foods as well as saving time and space.

Effect of heat treatment on activity of staphylococcal enterotoxins of type A, B, and C in milk

Intoxication by staphylococcal enterotoxins (SE) is among the most common causes of food-poisoning outbreaks resulting from the consumption of raw milk or products made thereof. The aim of our study was to analyze the thermal stability of SE and evaluate the inactivation of SE types A, B, and C (SEA, SEB, SEC) by autoclaving at 100°C, 110°C, and 121°C. Milk samples were inoculated with 38 Staphylococcus aureus strains that possessed the ability to produce SEA, SEB, or SEC and incubated at 37°C for 24 h. This incubation was followed by heat treatment at 100°C, 110°C, or 121°C for 3 min. Samples were analyzed by Staph. aureus plate count method on Baird-Parker agar and specifically for the presence of SE. An enzyme-linked immunofluorescent assay (ELFA) on a MiniVIDAS analyzer (bioMérieux, Marcy l'Étoile, France) was used to detect SE, which were determined semi-quantitatively based on test values. The obtained results were analyzed by means of nonparametric statistical methods. All samples (100%; 38/38) were SE-positive before heat treatment, and the positivity rates decreased after heat treatment at 100°C, 110°C, and 121°C to 36.8% (14/38), 34.2% (13/38), and 31.6% (12/38), respectively. The rates of positive samples differed between SEA, SEB, and SEC producers: SEA was detected in the highest amounts both before and after heat treatment. The amount of SE (expressed as test values) decreased significantly after heat treatment. Comparing amounts of SE in positive and negative samples before and after heat treatment, we can conclude that the success of SE inactivation depends on the amount present before heat treatment. The highest amount of SE and the highest rate of SE-positive samples after all heat treatments were found in samples with strains producing SEA. For SEB and SEC, lower amounts of enterotoxin were present and were inactivated at 100°C. Although temperatures of 100°C, 110°C, and 121°C may inactivate SE in milk, the key measures in prevention of staphylococcal enterotoxicosis are avoiding initial contamination of milk by Staph. aureus, promoting consumption of heat-treated milk, and preventing disruption of the cold chain during milk production and processing.

The Frequency of Staphylococcus aureus Classical Enterotoxin Genes in Raw Milk Samples in Zanjan, Iran

Background: Staphylococcus aureus is one of the major causes of food poisoning. Since milk is a nutritious source of proteins and vitamins, it could provide the optimal conditions for the growth of several bacterial pathogens, such as S. aureus. The present study aimed to assess the frequency of Staphylococcus aureus classical enterotoxin genes in raw milk samples in Zanjan, Iran. Methods: In total, 82 bovine, unpasteurized milk samples were collected from the dairy farms in various rural areas in Zanjan, Iran. The isolation and identification of S. aureus were performed using the Baird-Parker agar, routine biochemical tests, and polymerase chain reaction (PCR) targeting the S. aureus-specific femA gene. In addition, staphylococcal enterotoxin genes (e.g., sea, seb, sec, sed, and see) were assessed using PCR. Results: Following the appearance of yellow colonies with yellow zones on Mannitol salt agar, 21 S. aureus isolates (25.6%) were detected. In total, 80.9% of the isolates were positive for the presence of SE genes, and the most frequent SE gene was sea (88.2%), followed by see (58.8%), and seb (52.9%). Furthermore, 76.5% of the isolates had two or more SE genes simultaneously. Conclusion: According to the results, the presence of enterotoxigenic S. aureus in the studied raw milk samples confirmed the possible risk posed on the public health. Therefore, it is recommended that the quality of dairy product quality programs be optimized in order to intensify the sanitary inspection of these products.

Prevalence of Community-Associated Methicillin-Resistant Staphylococcus aureus in Oral and Nasal Cavities of 4 to 13-year-old Rural School Children: A Cross-sectional Study.

Aim:This study aimed to investigate the oral and nasal prevalence of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) in 4 to 13-year-old rural schoolchildren.Materials and Methods:A total of 100 children aged 4 to 13 years were randomly selected and divided into ten groups based on their age (Group 1 = 4-year-old children to Group 10 = 13-year-old children). From each participating child, sampling was done from the anterior nares and dorsum of the tongue. All samples were inoculated into Baird–Parker agar medium and HiCrome™ MeReSa agar medium for the isolation of SA and MRSA. Both the culture plates were checked for the presence of SA and MRSA and overall SA and MRSA carriage. The distribution of SA and MRSA was evaluated. Descriptive statistics were performed using SPSS software (version 17.0).Results:Overall SA in 4–13 years’ age group was 47%, while CA-MRSA was 35%. On the tongue, 16 children had concomitant MRSA and SA, while only 23.8% (n = 20) of the children comprised the presence of SA when MRSA was absent (P < 0.001). In the nasal cavity, 30 children had concomitant MRSA and SA, while only 21.4% (n = 15) of the children had the presence of SA when MRSA was absent (P < 0.001). When tongue and nose were assessed, 11 children encompassed concomitant MRSA and SA, while only 16.9% (n = 13) of the children had the presence of SA when MRSA was absent in both sites (P < 0.001).Conclusion:A significant relation was found between nasal SA and CA-MRSA carriage, with oral SA and CA-MRSA carriage. The study concludes that oral cavity is possibly as important as the nasal area as a zone of SA and MRSA. Dentists dealing with pediatric population should take proper precautions to prevent cross contamination of SA and MRSA in the dental clinic.

Prevalence of Enterotoxigenic Staphylococcus aureus and Shiga Toxin Producing Escherichia coli in Fish in Egypt: Quality Parameters and Public Health Hazard.

This study investigated the prevalence of Staphylococcus aureus enterotoxin genes and shiga toxin -producing Escherichia coli (STEC) in fish and evaluated quality parameters of examined fish. A total of 150 fish samples belonging to 6 species (25/each species) were cultured on Baird-Parker agar and eosin methylene blue agar. Staphylococcal enterotoxin genes and virulence genes (stx1, stx2, and eaeA genes) in E. coli serotypes were determined by multiplex PCR. Aerobic plate count (APC), Enterobacteriaceae count, coliform count, and Pseudomonas count were performed. Also, levels of total volatile base nitrogen and histamine in fish were determined. The prevalence of S. aureus ranged from 4% to 36% and count from 2 to 4 log10CFU/g. The sed, sea, and seb genes in S. aureus isolates were detected with percentages of 40%, 26.6%, and 20%, respectively. The E. coli serotype O26 carried stx1, stx2, and eaeA. The APCs, Enterobacteriaceae counts, and Pseudomonas counts ranged from 5.1 to 7.2, from 2.01 to 3.9, and from 2.1 to 3.1 log10 CFU/g, respectively. The most probable number (MPN) of coliform ranged from 1.3 to 3.6 log10/g. Levels of total volatile basic nitrogen and histamine ranged from 29.2 to 12.2 and from 0.6 to 4.6 mg/100 g, respectively. Also, the value of thiobarbituric acid was highly significant (1.1 ± 0.084 mg MDA/kg) in Trachurus mediterraneus samples compared with those levels obtained from other fish species. Our findings concluded that those fish species could constitute a public health hazard as fish are reservoirs for enterotoxigenic S. aureus and Shiga toxin producing E. coli strains. This study highlighted the importance of screening of fish for enterotoxigenic S. aureus strains and STEC isolates, and also assessing the quality parameters of fish.

Approaches for enhancing in situ detection of enterocin genes in thermized milk, and selective isolation of enterocin-producing Enterococcus faecium from Baird-Parker agar

Enterococci are naturally selected for growth in thermized ewes'/goats' milk mixtures used for traditional cooked hard cheese processing in Greece. A culture-independent PCR-based approach was applied to detect the presence of enterocin-encoding genes in naturally culture-enriched thermized milk (TM). Portions of TM (63 °C, 30 s) collected from a commercial cheese plant before addition of starters were fermented at 37 °C for 48 h to facilitate growth of indigenous enterococci. The multiple enterocin-producing (m-Ent+) Enterococcus faecium KE82 and the nisin A-producing Lactococcus lactis subsp. cremoris M104 served as bacteriocin-positive inocula in separate TM treatments. The PCR results revealed a constant presence of the enterocin A, B and P genes in TM fermented naturally at 37 °C. Eleven out of 42 (26.2%) lactic isolates from the enriched TM cultures without inoculation were Ent+ E. faecium assigned to three biotypes. Biotype I (4 isolates) included single entA possessors, whereas biotype II (5 isolates) and biotype III (2 isolates) were m-Ent+ variants profiling entA-entB-entP and entA-entB genes, respectively. Biotype II displayed the strongest antilisterial activity in vitro. Surprisingly, 85.7% (6/7) of the m-Ent+ E. faecium were selectively isolated from Baird-Parker agar, reflecting their natural resistance to 0.01% tellurite contained in the egg yolk supplement. No cytolysin-positive E. faecalis or other Ent+ Enterococcus spp. were isolated. In conclusion, commercially thermized Greek milk is a natural pool or ‘reservoir’ of antagonistic Ent+ or m-Ent+ E. faecium strains that can be easily detected and recovered by applying this PCR-based approach to naturally fermented milks or cheese products.

Biofilm Formation by Coagulase-Positive Staphylococcus aureus Isolated from Mozzarella Cheese Elaborated with Buffalo Milk and its Effect on Sensitivity to Sanitizers

Background: The buffalo milk mozzarella cheese is a new product in the market, with high consumer acceptance and excellent prospects for trade. The cheese is rich in nutrients, which favors the proliferation of microorganisms that can cause food-borne diseases in the consumer. Staphylococcus aureus can cause gastro-enteritis in humans by the production of enterotoxins in food. One problem that may hinder the elimination of undesirable microorganisms in the food industry is the formation of biofilms. The objective of this study was to determine the effect of biofilm formation by Staphylococcus aureus isolated from buffalo mozzarella cheese on sensitivity to sanitizers.Materials, Methods &amp; Results: Fifty samples of buffalo mozzarella cheese were analyzed to investigate the presence of S. aureus. The isolates were obtained through microbiological analysis and identified by PCR. The similarity of the strains was compared through rep-PCR. The distinct strains were tested for biofilm formation in microtiter plates. Soy Tripticase Broth (TSB) was placed in each well of the microtiter plate and overnight cultures of each strain was added. Wells without bacterial culture were used as controls. A villous cap was then placed on the plate and incubated for 48 h at 37°C. During incubation, the biofilms formed on the surface of the villi of the caps. For quantification of biofilm formation, material that remained attached to the cap was stained with crystal violet, the stained biofilm was extracted and the OD570 of each well was measured. Each strain was classified as non-biofilm forming, weak forming, moderately formed or formative strong. Strong forming and non-biofilm forming strains were tested on high density polyethylene, stainless steel and glass surfaces. Plates of 4 cm² of the different materials were placed in TSB where the culture of each isolate was inoculated separately. At each 48 h incubation the plates were washed to remove unbound cells and re-inserted into TSB without the inoculum. After five replicates of the procedure, sterile swabs were passed over the entire surface of each plate for counting in Baird-Parker agar. They were also tested for sensitivity to sodium hypochlorite and iodine after biofilm formation. The biofilm plates were immersed in flasks containing sanitizers, where they remained for 10 min. At the established contact time, the plates were immersed in neutralizing solution for 30 s. After washing with PBS, a sterile swab was passed on the surface of each plate and counts on Baird-Parker agar were performed. The bands profiles obtained on rep-PCR were identical when compared to isolates from the same sample, indicating that each sample was contaminated with only one S. aureus strain. From the twenty S. aureus strain identified, two isolates were classified as strong biofilm formers, seven as moderate formers, ten weak formers and one as non-biofilm builder. The two strong forming strains produced biofilm on the three surfaces tested. The application of sodium hypochlorite and iodine sanitizers promoted a reduction of approximately 2 log bacterial populations on all surfaces of both the biofilm and non-forming strains.Discussion: Most strains of S. aureus isolated from buffalo milk mozzarella cheese have the ability to form biofilm on the surfaces of equipment and utensils that have stainless steel, glass or high density polyethylene components. Although biofilm forming strains are no longer resistant to sanitizers sodium hypochlorite and iodine than non-forming sanitizers, they reach higher concentrations in the biofilm, resulting in larger bacterial populations remaining after application of the sanitizers. These results support the recommendation that the good hygienic practices adopted by industries processing buffalo milk mozzarella cheese should include specific measures to control the Staphylococcus aureus contamination.

MALDI-TOF mass spectrometry for the identification of Serbian artisanal cheeses microbiota

The study was carried out on 110 bacterial isolates obtained from Baird-Parker agar plates (n = 47) and de Man, Rogosa, Sharpe agar plates with added sorbic acid (n = 63), which were analyzed by MALDI-TOF mass spectrometry. Among 47 non-staphylococcal Baird-Parker agar isolates, Macrococcus caseolyticus was the most abundant (n = 37), followed by Enterococcus faecalis (n = 6). Corynebacterium aurimucosum, Corynebacterium glutamicum, Cronobacter sakazakii, and Enterococcus faecium were also identified. Among 63 non-Lactobacillus isolates, 18 isolates were identified as Leuconostoc mesenteroides, 17 as Enterococcus faecalis, 14 as Lactococcus lactis, 5 as Enterococcus durans, 5 as Lactococcus garvieae, 2 as Leuconostoc pseudomesenteroides, and 2 as Pediococcus pentosaceus. Enterococcus faecalis was the only species obtained from both types of media. All isolates were successfully identified to the species level. According to the results of this study, MALDI-TOF mass spectrometry proved to be useful tool for the identification of selected lactic acid bacteria and Baird-Parker agar isolates from artisanal cheeses.

Diversity of livestock associated methicillin-resistant Staphylococcus aureus.

To evaluate the diversity and molecular characteristics of livestock associated methicillin-resistant Staphylococcus aureus in livestock animals, food of animal origin and the environment in the Czech Republic. After having been primarily enriched in buffered peptone water, the samples were cultured on Baird-Parker agar. Presumptive colonies were sub-cultured to blood agar and assessed morphologically. Furthermore, presumptive Staphylococcus aureus colonies were confirmed by MALDI-TOF. Multiplex PCR, spa-typing, and MLST have been used to characterize the strains. Each mecA-positive Staphylococcus aureus isolates were examined against 14 different antimicrobials by using disk diffusion method. In this study, 13 different spa-types belonging to five sequence types (ST) were detected. Ninety four percent of tested strains belonged to CC/ST398 for which t011, t034, t2123 and t2346 were the vast major spa-types. In addition, non-ST398 clones such as CC1(t127), ST5(t3598), ST8(t064) and ST361(t315) were detected, which are known as human associated clones. The diversity of livestock associated methicillin-resistant Staphylococcus aureus has grown, and detecting lineages of human origin in animals and vice-versa becomes more common. Thus, livestock animal and its products will be a potential for the evolvement of methicillin-resistant Staphylococcus aureus in human population. Monitoring of pigs as well as other food-producing animal species and their products is therefore recommended.

Molecular characterization of antibiotic-resistant Staphylococcus aureus from livestock (bovine and swine).

Aim:The aim of this study was to figure the prevalence, phenotypic and genotypic antibiotic resistance (AR) pattern of Staphylococcus aureus isolated from bovine and swine nares.Materials and Methods:Colonies with typical morphology on Baird-Parker agar supplemented with egg-yolk tellurite emulsion were selected and biochemically/genotypically identified as S. aureus. These strains were further subjected to epsilometer test for their sensitivity to various clinically important antibiotics and antibiotic susceptibility testing for amoxicillin/clavulanic acid, and double-disk diffusion testing was performed by the standard disc diffusion method following CLSI guidelines. S. aureus strains were also tested for the presence of AR genes, viz., blaZ, mecA, aacA-aphD, erm (ermA, ermB, ermC), tet (efflux genes tetK and tetL, tetM and tetO of the ribosomal protection family), and vanA.Results:The nasal cavities of 17 out of 47 randomly selected bovine and 20 out of 28 randomly selected swine were positive for S. aureus, representing the prevalence of 36.2% (95% confidence interval [CI]: 22.5-49.9) and 71.4% (95% CI: 54.7-88.1), respectively. Most of the S. aureus strains showed higher resistance to penicillin (94.6%, minimal inhibitory concentration [MIC] ≥1.5 µg/ml) followed by ciprofloxacin (56.7%, MIC ≥32 µg/ml) and tetracycline (18.9%, MIC ≥32 µg/ml). About 10-15% of the strains were resistant to gentamicin (MIC 16 µg/ml) and oxacillin (MIC 6-8 µg/ml). None of the strains were resistant to vancomycin (MIC 0.25-1.5 µg/ml). In this study, 32.4% strains were resistant to three or more than three antibiotics and prevalence of this multi-drug resistant S. aureus was 45% (95%CI: 26.6-63.4) and 17.6% (95%CI: 6.7-28.5) in swine and bovine nasal samples, respectively. Four strains from pigs were borderline oxacillin-resistant S. aureus MIC 6-8 µg/ml, but none were mecA positive. Two of these strains were β-lactamase hyperproducers. Among the resistance genes blaZ, tetK, tetL, tetM, ermB, and aacA-aphD were found.Conclusion:Our results demonstrated the absence of mecA and pvl gene, but the presence of multi-drug resistant S. aureus in the nares of healthy animals which has a potential to spread in a community.

English

This paper identified the bacterial flora composition and its influence on the microbiological spoilage process in economically important fish species: Filhote (Brachyplatystoma filamentosum) and Dourada (Brachyplatystoma rousseauxii) in the Amazon region. Microbiological characterization was performed: counts of total mesophilic aerobic bacteria, psychrotrophic bacteria and coliforms at 35 and 45°C, respectively. Bacteria were also isolated through seeding in agar surface using Violet Red Bile Glucose (VRBG) for enterobacteria strains and Baird-Parker Agar with Egg-Yolk Tellurite for Staphylococcus species, both with incubation at 36°C for 48 h. The bacteria isolated were identified using the API 20E kit (Enterobacteria), and Gram-positive bacteria with API Staph (Staphylococci). Finally, the lag phase for strain growth was measured using spectrophotometry readings (620 nm) at different temperatures (10, 15 and 37°C). The mesophilic aerobic bacteria counts for fresh fish samples ranged from 5.21 to 7.64. The count ranges of psychrotrophic aerobic bacteria found were 5.11 – 6.91 log CFU/g. They also had an average score above 103 MPN/g for total coliforms. The most predominant were Hafnia alvei, Pseudomonas luteola, Staphylococcus xylosus and Staphylococcus lugdunensis. When subjected to temperature of 10°C, the strains achieved growth after 6 h (p<0.05). Key words: Spoilage, catfish, Brachyplatystoma filamentosum, Brachyplatystoma rousseauxii.

English

Staphylococcus aureus is an important pathogen that can cause Staphylococcal Food Poisoning (SFP). Milk and dairy products are frequently contaminated by this bacterium. In this study, 310 samples (168 bovine raw milk and 142 dairy products) were collected in the Tigray region of Northern Ethiopia, with the objective of detection and enumeration of S. aureus and other Staphylococcus species. Baird-Parker agar for isolation and 16S rRNA gene sequencing for species identification were employed. S. aureus was found in 38.7% of samples with mean count of 4.35 +/- 0.97 log 10 CFU ml -1. The prevalence of S. aureus was significantly greater in raw milk samples (47%) than in dairy products (28.8%). Of all S. aureus positive samples, 34.2% contained &gt;5 log10 CFU ml-1. Samples from cafeterias and restaurants showed the greatest prevalence of S. aureus (P&lt;0.05) compared to other sampling points. Samples from small-scale dairies were twice more likely to be contaminated by S. aureus than traditionally managed dairies (P&lt;0.05, OR=2.0). Coagulase-negative staphylococci (CNS) were found in 51.6% of the samples, with mean count of 6.0&plusmn;1.21 log10 CFU ml-1. Ten species of CNS were identified and S. epidermidis (36.13%) was the most frequent. The frequency of isolation found in this study indicates that S. aureus and other Staphylococcus spp. may impose public health hazard in dairy products. Therefore, further studies on the enterotoxigenic potential of the isolates, and molecular epidemiology to trace the sources of the contamination are recommended. &nbsp; Key words: Coagulase negative staphylococcus (CNS), dairy products, Ethiopia, Staphylococcus aureus, raw milk.