Occurrence of Mycobacterium bovis and non-tuberculous mycobacteria (NTM) in raw and pasteurized milk in the northwestern region of Paraná, Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Clonal profile, virulence and resistance of Staphylococcus aureus isolated from sheep milk

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Bacteriocinogenic and virulence potential of Enterococcus isolates obtained from raw milk and cheese

Aims To provide molecular and phenotypical characterization of Enterococcus isolates obtained from raw milk and cheese, regarding their bacteriocinogenic and virulence activity. Methods and Results Forty-three bacteriocinogenic enterococci isolates were identified by 16s rDNA, fingerprinted by RAPD-PCR analysis and tested by PCR for the presence of genes for lantibiotics (lanM, lanB and lanC) and enterocins (entA, entB, entP, entL50AB and entAS48) and by phenotypical methods for bacteriocin production and inhibitory spectrum. Also, the virulence of the isolates was evaluated by PCR for genes gelE, hyl, asa1, esp, cylA, efaA, ace, vanA, vanB, hdc1, hdc2, tdc and odc and by phenotypical tests for gelatinase, lipase, DNAse and a- and beta-haemolysis. Most isolates (93.0%) harboured at least one lantibiotic or enterocin gene and were positive for several tested virulence genes, mainly asa1 (100%), gelE (93.0%) and efaA (83.7%). 53.5% of the isolates presented beta-haemolysis. Conclusions Enterococcus spp. isolates presented an interesting potential application for food preservation because of bacteriocin production; however, virulence-related genes were identified in all RAPD profiles. Significance and Impact of the Study The study demonstrated the contradictory characteristics of the tested Enterococcus isolates: they presented a good potential for application in food biopreservation but contained several virulence factors.

Milk hygienic practices and occurrence of Staphylococcus aureus and Escherichia coli O157:H7 in small-scale dairy farms in Sao Paulo, Brazil

The objective of this study was to assess the relationship between somatic cell counts (SCC), the use of different milking practices, and the occurrence of Staphylococcus aureus and Escherichia coli O157:H7 in 42 small-scale dairy farms located in the state of Sao Paulo, Brazil. S. aureus and E. coli O157:H7 were isolated in the milk from dairy cows with low (< 200,000 cells/ml) and high SCC (>200,000 cells/ml), although no effect of SCC (p > 0.05) was observed on the incidence of the bacteria in raw milk. The use of disposable gloves during milking reduced S. aureus counts in milk (p < 0.05), but did not affect the occurrence of E. coli O157:H7. The other milking practices evaluated (closed milking system, use of pre- and post-dipping, mastitis diagnosis by strip cup test, and disinfection of teat cups) did not affect (p < 0.05) the occurrence of S. aureus or E. coli O157:H7 in raw milk. Results indicate the need for effective educational programs addressed to prevent the contamination of milk with S. aureus and E. coli O157:H7 in Brazilian small-scale dairy farms.

Nonculture-based identification of bacteria in milk by protein fingerprinting

Traditional methods for bacterial identification include Gram staining, culturing, and biochemical assays for phenotypic characterization of the causative organism. These methods can be time-consuming because they require in vitro cultivation of the microorganisms. Recently, however, it has become possible to obtain chemical profiles for lipids, peptides, and proteins that are present in an intact organism, particularly now that new developments have been made for the efficient ionization of biomolecules. MS has therefore become the state-of-the-art technology for microorganism identification in microbiological clinical diagnosis. Here, we introduce an innovative sample preparation method for nonculture-based identification of bacteria in milk. The technique detects characteristic profiles of intact proteins (mostly ribosomal) with the recently introduced MALDI SepsityperTM Kit followed by MALDI-MS. In combination with a dedicated bioinformatics software tool for databank matching, the method allows for almost real-time and reliable genus and species identification. We demonstrate the sensitivity of this protocol by experimentally contaminating pasteurized and homogenized whole milk samples with bacterial loads of 10(3)-10(8) colony-forming units (cfu) of laboratory strains of Escherichia coli, Enterococcus faecalis, and Staphylococcus aureus. For milk samples contaminated with a lower bacterial load (104 cfu mL-1), bacterial identification could be performed after initial incubation at 37 degrees C for 4 h. The sensitivity of the method may be influenced by the bacterial species and count, and therefore, it must be optimized for the specific application. The proposed use of protein markers for nonculture-based bacterial identification allows for high-throughput detection of pathogens present in milk samples. This method could therefore be useful in the veterinary practice and in the dairy industry, such as for the diagnosis of subclinical mastitis and for the sanitary monitoring of raw and processed milk products.

Evaluation of different selective media for enumeration of probiotic micro-organisms in combination with yogurt starter cultures in fermented milk

The aim of this study was to assess selective plating methodologies for the enumeration and identification of Streptococcus thermophilus, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus acidophilus, Lactobacillus rhamnosus and Bifidobacterium animalis ssp. lactis in fermented milks. Seven agar media (MRS with added sorbitol, clindamycin or vancomycin, acidified MRS, RCA with added aniline blue and dicloxacilin, M17 and ST) were evaluated. The results showed that RCA dicloxacilin agar was suitable for the selective enumeration of B. animalis ssp. lactis in fermented milk. Either MRS (acidified) or M17 agar could be used for enumeration of L. delbrueckii ssp. bulgaricus and S. thermophilus, respectively. MRS media containing antibiotics were effective for the enumeration of the probiotic organisms (L. rhamnosus and L. acidophilus) inoculated in fermented milks.

Culture medium of diluted skimmed milk for the production of nisin in batch cultivations

Nisin is a promising alternative to chemical preservatives for use as a natural biopreservative in foods. This bacteriocin has also potential biomedical applications. Lactic acid bacteria are commonly cultivated in expensive standard complex media. We have evaluated the cell growth and nisin production of Lactococcus lactis in a low-cost natural medium consisting of diluted skimmed milk in a 2-L bioreactor. The assays were performed at 30 degrees C for 56 h, at varying agitation speeds and airflow rates: (1) 200 rpm (no airflow, and airflow at 0.5, 1.0 and 2.0 L/min); (2) 100 rpm (no airflow, and airflow at 0.5 L/min). Nisin activity was evaluated using agar diffusion assays. The highest nisin concentration, 49.88 mg/L (3.3 log AU/mL or 1,995.29 AU/mL), was obtained at 16 h of culture, 200 rpm and no airflow (k(L)a = 5.29 x 10(-3)). These results show that a cultivation medium composed of diluted skimmed milk supports cell growth to facilitate nisin biosynthesis.

Antibiotic resistance in Lactococcus species from bovine milk: presence of a mutated multidrug transporter mdt(A) gene in susceptible Lactococcus garvieae strains

A total of 72 Lactococcus strains (41 Lactococcus lactis and 31 Lactococcus garvieae) isolated from bovine milk were tested for susceptibility to 17 antibiotics and screened for the presence of antibiotic resistance genes using a microarray. Resistance to tetracycline, clindamycin, erythromycin, streptomycin, nitrofurantoin were found. The tetracycline-resistant L. garvieae and L. lactis harbored tet(M) and tet(S). L. lactis that were resistant to clindamycin were also resistant to erythromycin and possessed the erm(B) gene. The multidrug transporter mdt(A), originally described in L. lactis, was detected for the first time in L. garvieae and does not confer decreased susceptibility to erythromycin nor tetracycline in this species. Mdt(A) of L. garvieae contains one mutation in each antiporter motif C, which is known to play an essential role in drug efflux antiporters. This suggests that the mutations found in the C-motifs of Mdt(A) from L. garvieae may be responsible for susceptibility. The study revealed the presence of antibiotic resistance genes in non-pathogenic and pathogenic lactococci from bovine milk, including a mutated multidrug transporter in L. garvieae.

Role of milk and meat products as vehicles for antibiotic-resistant bacteria

This subject is reviewed under the following headings: Microbial contamination of raw meat and raw milk; Antibiotic resistance of food-borne pathogens; Antibiotic resistance of commensal and potentially pathogenic bacteria as a new threat in food microbiology; Antibiotic-resistant staphylococci in fermented meat and [in] milk products; Antibiotic-resistant Enterococcus sp. in fermented meat and [in] milk products; Enterococci in farm animals and meat; Enterococci in fermented food; Molecular characterization of resistance of food-borne enterococci; and Further ecological and epidemiological considerations of resistant live bacteria in food. It is concluded that further research is needed, particularly into the possible transfer of the resistance of bacteria consumed in meat or milk products to the indigenous bacteria of the human consumer.

Health-promoting properties of bioactive peptides derived from milk proteins in infant food : a review

Peer reviewed

Sensitive and specific detection of viable Mycobacterium avium subsp. paratuberculosis in raw milk by the Peptide-mediated magnetic separation (PMS)-phage assay

This study describes further validation of a previously described Peptide-mediated magnetic separation (PMS)-Phage assay, and its application to test raw cows’ milk for presence of viable Mycobacterium avium subsp. paratuberculosis (MAP). The inclusivity and exclusivity of the PMS-phage assay were initially assessed, before the 50% limit of detection (LOD50) was determined and compared with those of PMS-qPCR (targeting both IS900 and f57) and PMS-culture. These methods were then applied in parallel to test 146 individual milk samples and 22 bulk tank milk samples from Johne’s affected herds. Viable MAP were detected by the PMS-phage assay in 31 (21.2%) of 146 individual milk samples (mean plaque count of 228.1 PFU/50 ml, range 6-948 PFU/50 ml), and 13 (59.1%) of 22 bulk tank milks (mean plaque count of 136.83 PFU/50 ml, range 18-695 PFU/50 ml). In contrast, only 7 (9.1%) of 77 individual milks and 10 (45.4%) of 22 bulk tank milks tested PMS-qPCR positive, and 17 (11.6%) of 146 individual milks and 11 (50%) of 22 bulk tank milks tested PMS-culture positive. The mean 50% limits of detection (LOD50) of the PMS-phage, PMS-IS900 qPCR and PMS-f57 qPCR assays, determined by testing MAP-spiked milk, were 0.93, 135.63 and 297.35 MAP CFU/50 ml milk, respectively. Collectively, these results demonstrate that, in our laboratory, the PMS-phage assay is a sensitive and specific method to quickly detect the presence of viable MAP cells in milk. However, due to its complicated, multi-step nature, the method would not be a suitable MAP screening method for the dairy industry.

MICROBIOLOGICAL ANALYSIS OF TRADITIONALLY FERMENTED MILK SOLD IN KINIGI SECTOR OF MUSANZE DISTRICT IN RWANDA

A research work entitled: “Microbiological analysis of traditionally fermented milk (Ikivuguto) sold in Kinigi Sector of Musanze District,” was carried out at Higher Learning Institution of Applied Sciences (INES-Ruhengeri) Laboratory of Microbiology located near Volcanoes in the Northern Province of Rwanda. The main objective of this work was to determine the microbiological quality of traditionally fermented milk, which is consumed by Kinigi Center local people. The hypothesis was to analyze if traditionally fermented milk commercialized in Kinigi restaurants contained pathogenic bacteria such as fecal coliforms and Escherichia coli , in addition to staphylococci and yeasts. Milk samples were collected from Kinigi sector and examined in the microbiology laboratory in order to assess the microbiological quality and safety of traditionally fermented milk in rural areas. The samples were analyzed qualitatively and quantitatively for the microbes found in fermented milk sold in Kinigi Center, and the results were as follows: 7.21x107 CFU/ml for total counts; 3.89x107 CFU/ml for Lactobacillus ; 2.77x107 CFU/ml for yeasts; 1.196x105 CFU/ml for total coliforms; 9.63x104 CFU/ml for fecal coliforms and 8.92x103 CFU/ml for staphylococci. Biochemical tests were carried out and the results showed that identified pathogens were E. coli, Providencia alcalifaciens , and the staphylococci group. It was found that fermented milk contained genera and species of Staphylococcus haemolyticus , Staphylococcus aureus , Staphylococcus intermedius , Staphylococcus xylosus and Staphylococcus saprophyticus . Findings showed that the commercial milk samples were cross-contaminated by different pathogens from environment. These contaminations could have been due to improper handling, presence of flies, soil erosion, dust from atmosphere, as well as contaminated milk vessels or pots, stirrers and unpasteurized water. It was concluded that local farmers and milk retailers did not adhere to required hygienic conditions for milk safety. In this regard, the sold traditional fermented milk does not meet health and safety standards because people did not respect good manufacturing practices. The hypothesis and main objective were confirmed, because traditionally fermented milk of Kinigi was cross-contaminated before consumption. Thus, it would be better to train farmers in the areas of product hygiene, sanitation and safety during milking, processing and marketing.

MiLK: Transforming everyday places into dynamic learning spaces with mobile and web technologies

This paper explores how game authoring tools can teach processes that transform everyday places into engaging learning spaces. It discusses the motivation inherent in playing games and creating games for others, and how this stimulates an iterative process of creation and reflection and evokes a natural desire to engage in learning. The use of MiLK at the Adelaide Botanic Gardens is offered as a case in point. MiLK is an authoring tool that allows students and teachers to create and share SMS games for mobile phones. A group of South Australian high school students used MiLK to play a game, create their own games and play each other’s games during a day at the gardens. This paper details the learning processes involved in these activities and how the students, without prompting, reflected on their learning, conducted peer assessment, and engaged in a two-way discussion with their teacher about new technologies and their implications for learning. The paper concludes with a discussion of the needs and requirements of 21st century learners and how MiLK can support constructivist and connectivist teaching methods that engage learners and will produce an appropriately skilled future workforce.

MILK : the Mobile Informal Learning Kit. Collaborating to design successful mobile learning applications

This paper suggests ways for educators and designers to understand and merge priorities in order to inform the development of mobile learning (m-learning) applications that maximise user experiences and hence learning opportunities. It outlines a User Experience Design (UXD) theory and development process that requires designers to conduct a thorough initial contextual inquiry into a particular domain in order to set project priorities and development guidelines. A matrix that identifies the key contextual considerations namely the social, cultural, spatial, technical and temporal constructs of any domain is presented as a vital tool for achieving successful UXD. The frame of reference provided by this matrix ensures that decisions made throughout the design process are attributable to a desired user experience. To illustrate how the proposed UXD theory and development process supports the creation of effective m-learning applications, this paper documents the development process of MILK (Mobile Informal Learning Kit). MILK is a support tool that allows teachers and students to develop event paths that consist of a series SMS question and answer messages that lead players through a series of checkpoints between point A and point B. These event paths can be designed to suit desired learning scenarios and can be used to explore a particular place or subject. They can also be designed to facilitate formal or informal learning experiences.