M.M.O.P. Cerqueira

A study of the enterotoxigenicity of coagulase-negative and coagulase-positive staphylococcal isolates from food poisoning outbreaks in Minas Gerais, Brazil

OBJECTIVES The purpose of this study was to identify enterotoxin genes from isolates of coagulase-negative staphylococci and coagulase-positive staphylococci obtained from dairy products, responsible for 16 outbreaks of food poisoning. METHODS From the pool of 152 staphylococcal isolates, 15 coagulase-negative and 15 coagulase-positive representatives were selected for this study. The 15 coagulase-negative isolates were tested for the presence of coa and femA genes, which are known to be characteristic of Staphylococcus aureus. After testing for enterotoxin genes by polymerase chain reaction (PCR), the 30 selected isolates were tested for the presence of toxin by immunoassay. RESULTS Seven of the coagulase-negative isolates amplified the coa gene and were subsequently reclassified as coagulase-positive. Twenty-one of 30 selected isolates had staphylococcal enterotoxin genes and most of these produced toxin as well. The most frequently encountered enterotoxin genes were sea and seb. Among eight coagulase-negative isolates, five had enterotoxin genes, all of which were found to have detectable toxin by immunoassay. CONCLUSIONS The results from this study demonstrate that coagulase-negative as well as coagulase-positive staphylococci isolated from dairy products are capable of genotypic and phenotypic enterotoxigenicity. Furthermore, these data demonstrate that PCR is a sensitive and specific method for screening outbreak isolates regardless of coagulase expression.

Contagem de Staphylococcus sp. e detecção de enterotoxinas estafilocócicas e toxina da síndrome do choque tóxico em amostras de leite cru refrigerado

In order to count and identify Staphylococcus sp., the detection of the Staphylococcal enterotoxins (SE) and toxic shock toxin syndrome (TSST-1), 80 raw milk samples cooled at 4°C and stored in bulk tanks for 48 hours in different farms from Minas Gerais State were analyzed. Staphylococcus sp. was observed in all samples and the counts varied from 1.0 × 105 to 2.5 × 107 CFU/ml (mean = 5.60 log CFU/ml; sd = 0.53 and CV = 9.46 %). A total of 436 strains of Staphylococcus were isolated and identified as S. aureus, S hyicus, S. epidermidis, S. intermedius, S. cohnii, S. sciuri, S. schleirferi and S. delphini. Strains showing identical biochemical profile, from the same sample, were grouped into a pool and them were induced to produce SE and TSST-1. The detection of toxins was made by the OSP (Optimum Sensivity Plate) method and the cellophane-over-agar technique. It was identified SEA, SEB, SEC, SED and TSST-1 in different percentages. From the 138 formed pools, 91 produced, at least, one or more toxin, including TSST-1. From the enterotoxigenic pools, 24.6% were coagulase positive, while 41.3% were negative. The presence of entorotoxigenic negative coagulase Staphylococcus strains isolated from milk samples is important in relation to public health.

Bactérias do acido láctico e leveduras associadas com o queijo-de-minas artesanal produzido na região da Serra do Salitre, Minas Gerais

Samples of milk, curd, cheese whey, and cheese were collected in 10 farms located at the region of Serra do Salitre, Minas Gerais state. These samples were studied in relation to their lactic acid bacteria and yeast populations. The diversity of lactic acid bacteria species was lower than the diversity of yeasts in these samples. The isolated lactic acid bacteria were Lactococcus lactis, Enterococcus spp., Enterococcus faecalis, and Streptococcus agalactiae; and the yeasts were Debaryomyces hansenii and Kluyveromyces lactis. Only the species Enterococcus spp., Enterococcus faecalis, and Leuconostoc mesenteroides showed an increase in their populations during the production of the artisanal cheese. Lactic acid bacteria and yeasts found in this study could be responsible by the sensorial characteristics of the artisanal cheese produced in the region of Serra do Salitre.

Presença de Staphylococcus spp. produtores de enterotoxinas e da toxina da síndrome do choque tóxico em manipuladores de queijo de cabra

A total of 167 strains of Staphylococcus isolated from nasal cavities, oropharynx, palm of hands and subunguial of two goats cheese handlers were collected. The strains were pooled (45) according to the species similarity and place of origin and tested for the production of enterotoxins (SE) A, B, C, D and toxic shock toxin syndrome (TSST-1). It was observed that 62.2% of the pools presented the capacity to produce, individually or in association, SEA (33.3%); SEB (46.7%); SEC (8.9%); SED (4.4%) and TSST-1 (4.4%). From the enterotoxigenic pools, 96.4% corresponded to species negative coagulase (Staphylococcus epidermidis and Staphylococcus cohnii). The capacity to produce SE and TSST-1 by Staphylococcus spp. strains isolated from food handlers reaffirms its hole on the transmission of food poisoning, beyond emphasizing the indispensable necessity of the adoption of satisfactory hygienic and sanitary procedures during the food manufacture.

Evolução anual da qualidade do leite cru refrigerado processado em uma indústria de Minas Gerais

The present study aimed to analyze the annual evolution of cooled raw milk quality, through analytical analysis of the data base of individual bulk milk tanks analysis, monthly computed from April/2002 to December/2008 at a dairy industry in Minas Gerais. The mean volume of milk in compliance with IN51 for total bacterial count for year 2008 increased from 74.3% in 2002 to 85.8% in 2008. Considering IN51 for year 2011, these values would be 30.5% and 46.6%, respectively. The mean volume of milk in compliance with IN51 for somatic cells count reduced from 93.5% in 2002 to 90.1% in 2008. Considering the legal standard for the year of 2011, these values would be 64.9 and 60.5%, respectively. In relation to the solid composition of milk, the mean volume of milk in compliance with IN51 for fat, protein and solids nonfat were 95.2%, 98.2% and 89.6%, respectively. The season variations affected the quality of the bulk tank cooled raw milk from bulk tanks. The implantation of a payment system for the quality of milk to the producer revealed to be efficient in its objective to improve the quality indexes of raw milk.

The neutrophil function and lymphocyte profile of milk from bovine mammary glands infected with Streptococcus dysgalactiae

Streptococcus dysgalactiae is a bacterium that accounts for a notable proportion of both clinical and subclinical intramammary infections (IMIs). Thus, the present study explores the function of milk neutrophils and the lymphocyte profile in mammary glands naturally infected with Streptococcus dysgalactiae. Here, we used 32 culture-negative control quarters from eight clinically healthy dairy cows with low somatic cell counts and 13 S. dysgalactiae-infected quarters from six dairy cows. Using flow cytometry, we evaluated the percentage of milk monocytes/macrophages and neutrophils, expression of CD62L, CD11b and CD44 by milk neutrophils, the levels of intracellular reactive oxygen species (ROS) production and phagocytosis of Staphylococcus aureus by milk neutrophils, and neutrophil viability. Furthermore, the percentages of B cell (CD21(+)) and T lymphocyte subsets (CD3(+)/CD4(+)/CD8(-); CD3(+)/CD8(+)/CD4(-); and CD3(+)/CD8(-)/CD4(-)), and the expression of CD25 by T milk lymphocytes (CD3(+)) and T CD4(+) milk cells were also assessed by flow cytometry using monoclonal antibodies. The present study showed a higher SCC and percentage of milk neutrophils, and a decrease in the percentage of milk monocytes/macrophages from S. dysgalactiae-infected quarters when compared to uninfected ones. We also observed a higher expression of CD11b by milk neutrophils and a tendency toward a decrease in neutrophil apoptosis rate in S. dysgalactiae-infected quarters. In addition, the S. dysgalactiae-infected quarters had higher percentages of milk T cells (CD3(+)) and their subset CD3(+)CD8(+)CD4(-) cells. Overall, the present study provided new insights into S. dysgalactiae IMIs, including distinct lymphocyte profiles, and a tendency toward an inhibition of apoptosis in milk neutrophils.

Interaction between bovine-associated coagulase-negative staphylococci species and strains and bovine mammary epithelial cells reflects differences in ecology and epidemiological behavior

Bacteria adherence seems to be an essential first stage for the internalization of bacteria into the cytoplasm of the host cell, which is considered an important virulence strategy enabling bacteria to occupy a microenvironment separated from host defense mechanisms. Thus, this study aimed to explore the difference in the capacity of 4 bovine-associated staphylococci species or strains to adhere to and internalize into bovine mammary epithelial cells (MEC). Three different isolates of coagulase-negative staphylococci (CNS) were used: one strain of Staphylococcus fleurettii isolated from sawdust and considered an environmental opportunistic bacterium, and 2 dissimilar Staphylococcus chromogenes isolates, one cultured from a heifers teat apex (Staph. chromogenes TA) and the other originating from a chronic intramammary infection (Staph. chromogenes IM). Also, one well-characterized strain of Staphylococcus aureus (Newbould 305) was used for comparison with a major mastitis pathogen. The CNS species and strains adhered to and internalized into MEC slower than did Staph. aureus. Still, we observed high variation in adhesion and internalization capacity among the different CNS, with Staph. chromogenes IM showing a greater ability to adhere to and internalize into MEC than the 2 CNS strains isolated from extramammary habitats. In conclusion, the 3 well-characterized bovine-associated CNS species and strains originating from distinct habitats showed clear differences in their capacity to adhere to and internalize into MEC. The observed differences might be related to their diversity in ecology and epidemiological behavior.

Detecção de Listeria monocytogenes pela técnica de PCR em leite contaminado artificialmente

The polymerase chain reaction (PCR) was used to detect Listeria monocytogenes in inoculated milk samples after selective enrichment. Samples of sterile skim milk and raw whole milk (with low, intermediate, and high counts of aerobic mesophilic microorganisms) were inoculated with several concentrations of L. monocytogenes. The results of PCR assays were compared to the results of culturing the samples using a standardized traditional method for isolation of L. monocytogenes. The pathogen was detected by PCR in Listeria Enrichment Broth (LEB) after 48h-incubation (sensitivity of 1CFU/mL) but not after 24h-incubation from the samples prepared with sterile skim milk. L. monocytogenes was not detected by PCR in LEB after 24 and 48h-incubation from the samples prepared with raw whole milk. Using the traditional method, the pathogen was detected in all experiments. However, sensitivity decreased in raw whole milk with high counts of aerobic mesophilic microorganisms (up to 7CFU/mL). Best results were obtained when PCR was done to identify presumptive L. monocytogenes colonies directly from Palcam and Oxford media, after the enrichment step. This procedure allowed reducing to a few hours the period of several days usually needed to obtain the final identification of L. monocytogenes using phenotypic tests.

Flow cytometric analysis: Interdependence of healthy and infected udder quarters

An important question about intramammary infections that is still debated in the literature is the independence or interdependence of the quarters of dairy cows. The present study sought to explore milk neutrophil function and the milk lymphocyte profile of uninfected quarters from uninfected and infected (one infected quarter per cow) udders to evaluate interdependence of the quarters. Thus, 32 (8 cows) and 18 (6 cows) uninfected quarters from uninfected and infected udders were used, respectively. Using flow cytometry, we evaluated the percentage of milk neutrophils and their expression of adhesion molecules L-selectin (CD62L), β2-integrin (CD11b), and an endothelial-selectin ligand (CD44); levels of intracellular reactive oxygen species (ROS); phagocytosis of Staphylococcus aureus by milk neutrophils; and neutrophil viability. Furthermore, we assessed the percentage of B-cell (CD21(+)) and T-lymphocyte subsets (CD3(+)/CD4(+)/CD8(-), CD3(+)/CD8(+)/CD4(-), CD3(+)/CD4(+)/CD25(-), CD3(+)/CD4(+)/CD25(+), and CD3(+)/CD4(-)/CD25(-)) using flow cytometry with monoclonal antibodies. The infected quarter did not affect somatic cell count or the percentage of neutrophils in the neighboring uninfected quarters. Furthermore, the infected quarter did not influence neutrophil viability, intracellular reactive oxygen species production, or phagocytosis of S. aureus by milk neutrophils. Conversely, the expression of adhesion molecules CD11b, CD62L, and CD44 by milk neutrophils differed between uninfected quarters from infected versus uninfected udders. The lymphocyte subsets did not differ between groups, except for a higher percentage of B cells in uninfected quarters from infected udders than in those from uninfected udders. Thus, our study strongly supports the hypothesis of interdependence of quarters based on the influence of infection on both the percentage of B cells and the expression of adhesion molecules by milk neutrophils in the neighboring uninfected quarters.

Detection of cheese whey in raw milk preserved with bronopol® through high performance liquid chromatography

High performance liquid chromatography was used in order to detect cheese whey in samples of raw milk preserved with Bronopol®. Six samples were collected and divided in 45 aliquots of 40mL. From these, 15 were used as control and stored frozen, 15 were added with Bronopol® and stored at 7oC, and the other 15 were added with Bronopol® and stored at 30oC. In all groups, five levels of cheese whey addition (0, 2, 5, 10, and 20%) were tested. The samples were submitted to high performance liquid chromatography on the 2nd, 4th, and 8th days of storage. A completely random design was used, following the factorial scheme (5x3x3) and the results were compared through the non-parametric Kruskal-Wallis test. There was no difference among the treatments (P>0.05), which allows the conclusion that raw milk preserved with Bronopol® may be used for the determination of cheese whey addition in milk through high performance liquid chromatography.