Detection by multiplex PCR of  Staphylococcus aureus ,  S. intermedius and  S. hyicus in artificially contaminated milk

Gandra, Eliézer Ávila; Fernandez, Maria Aparecida; Silva, Jorge Adolfo; Silva, Wladimir Padilha da

doi:10.1590/0103-8478cr20151391

Cited by 6 publications

(4 citation statements)

References 14 publications

(18 reference statements)

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“…Para identificação das espécies, o DNA dos isolados que apresentaram reação positiva para produção de coagulase foi extraído [23] e analisado pela reação em cadeia da polimerase (PCR) [9]. Para identificar isolados do mesmo isolado, estes foram analisados pela rep-PCR [20], utilizando o primer (GTG) 5 (GTGGTGGTGGTGGTG) [27], o qual apresenta elevado poder de discriminação para Staphylococcus spp.…”

Section: Obtenção E Identificação Dos Isoladosunclassified

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

Friedriczewski

Gandra

Conceição

et al. 2018

Acta Scientiae. Vet.

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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 & 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...

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Section: Obtenção E Identificação Dos Isoladosunclassified

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

Friedriczewski

Gandra

Conceição

et al. 2018

Acta Scientiae. Vet.

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“…Penggunaan multipleks PCR memberi keuntungan cepatnya hasil deteksi serovar yang berbeda diantara campuran kontaminan Salmonella, tetapi memberikan persentase hasil yang lebih rendah dibandingkan dengan simpleks PCR. Berdasarkan penelitian Gandra et al (2016), metode multipleks PCR menurunkan sensitivitas deteksi dibandingkan dengan metode simpleks PCR, karena pada multipleks PCR dapat terjadi kompetisi amplifikasi antara satu target gen dengan target gen lainnya sehingga amplifikasi tidak dapat berjalan dengan baik.…”

Section: Peningkatan Presentase Hasil Positif Metode Preenrichment Un...unclassified

SIMPLEKS DAN MULTIPLEKS PRE-ENRICHMENT-PCR UNTUK DETEKSI Salmonella Enteritidis DAN Typhimurium PADA KARKAS AYAM

Nurjanah¹,

Rahayu²,

Dewanti‐Hariyadi³

et al. 2021

jtip

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A PCR assay has been developed and applied to detect Salmonella contamination in chicken carcasses. However, a concentration fewer than 3 cells per gram lead to false-negative results due to difficulties in the DNA extraction. The objective of this study was to evaluate of the influence of pre-enrichment on the sensitivity of simplex and multiplex PCR methods the detection of for Salmonella spp., S. Enteritidis and S. Typhimurium in chicken carcasses. Artificial contamination was done using very low number of Salmonella Hadar, S. Enteritidis dan S. Typhimurium and pre-enrichment was carried out by 8 hours incubation in non-selective (BPW) medium. The results showed that simplex PCR could detect Salmonella spp., S. Enteritidis and S. Typhimurium at initial numbers of 2.3, 0.9 and 2.3 MPN/mL of cells in broth medium, respectively. A multiplex PCR could detect mixed culture of the three Salmonella serovars at an initial number of 0.73 MPN/mL of cells. When compared to non-enrichment treatment, simplex pre-enrichment-PCR gave an increase in the percentage of positive results in chicken carcasses (n= 12), from 75 to 100% for Salmonella spp., from 8 to 58% for S. Typhimurium, and from 58 to 75% for S. Enteritidis. Increasing in the positive percentage was also occurred at multiplex pre-enrichment-PCR, however the concentration of S. Enteritidis primer was not optimum for detection. Pre-enrichment step significantly increases the sensitivity of PCR-based assay for detection Salmonella.

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“…Most staphylococcal food intoxication outbreaks are associated with classical SE, sea-see; however, sea is the most widely implicated [7,8]. Several molecular techniques are used for the rapid, accurate detection of S. aureus isolates, including multiplex polymerase chain reaction (PCR) [9,10].…”

Section: Introductionmentioning

confidence: 99%

Molecular detection of Staphylococcus aureus enterotoxin genes isolated from mastitic milk and humans in El-Behira, Egypt

et al. 2021

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Background and Aim: Milk is a chief source of many nutrients. However, we must also bear in mind that it is a potential source for many cases of food poisoning. This study was conducted to investigate the prevalence of cow mastitis and evaluate the presence of enterotoxins and antibiotic resistance patterns in Staphylococcus aureus isolated from milk and contact humans in El-Behira Province, Egypt. Materials and Methods: A total of 680 milk samples from 170 cows and 86 human samples consisting of 43 hand swabs and 43 nasal swabs were analyzed. The milk samples were subjected to the California mastitis test. Results: The general occurrence was 23.1% (157/680) where 48 quarters had clinical mastitis and 109 had subclinical mastitis. Subsequently, S. aureus was isolated in Baird-Parker agar where typical and atypical colonies were selected and submitted to coagulase and complementary tests. Out of 48 samples of mastitic milk studied, 16 (33.3%) showed contamination by S. aureus whereas 109 samples of subclinical mastitis showed contamination in only 18 (16.5%). On the opposite hand, of the 86 human samples, 33 revealed S. aureus contamination, corresponding to 38.37% of the samples. Furthermore, multiplex polymerase chain reaction targeting nuc and the staphylococcal enterotoxin-encoding genes sea, seb, sec, sed, and see were performed after culture, revealing that 88.2% (30/34) of milk samples and 93.9% (31/33) of human samples were variably positive to those genes. Conclusion: The use of nuc gene based PCR is an accurate and rapid method for S aureus isolates detection. A high prevalence of multiple drug-resistant isolates of S. aureus recovered from both human and milk represents further evidence for possible veterinary hazards as well as public hazards, especially to those that consume milk from this region.

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Detection by multiplex PCR of Staphylococcus aureus , S. intermedius and S. hyicus in artificially contaminated milk

Abstract: Detection by multiplex PCR of Staphylococcus aureus, S. intermedius and S. hyicus in artificially contaminated milk.Ciência Rural, v.46, n.8, ago, 2016.

Cited by 6 publications

References 14 publications

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

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

SIMPLEKS DAN MULTIPLEKS PRE-ENRICHMENT-PCR UNTUK DETEKSI Salmonella Enteritidis DAN Typhimurium PADA KARKAS AYAM

Molecular detection of Staphylococcus aureus enterotoxin genes isolated from mastitic milk and humans in El-Behira, Egypt

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