scholarly journals MULTIPLEX-PCR FOR DETECTION OF β-LACTAM RESISTANCE IN Staphylococcus spp.

2019 ◽  
Vol 6 (2) ◽  
pp. 262-275
Author(s):  
Giovana Hashimoto Nakadomari ◽  
Amanda Carmen Charalo ◽  
Ana Claudia Lemes Pavan ◽  
Vanessa Kelly Capoia Vignoto ◽  
Ricardo Antonio Pilegi Sfaciotte ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Resistance Genes ◽  
Internal Control ◽  
Meca Gene ◽  
Simultaneous Detection ◽  
Rrna Gene ◽  
16S Gene ◽  
Internal Reaction ◽  
Two Samples ◽  
Staphylococcus Spp

A Staphylococcus Multiplex PCR system was developed for the simultaneous detection of the mecA, mecC, blaZ (resistance genes of b-lactam resistance) and PVL (pathogenicity factor gene), associated with an internal reaction control with the 16S rRNA gene. There were used primers described in the literature with and without modification and designed primers to standardize the hybridization and amplification temperature of distinct bands with 139 bp (mecC), 228 bp (16S), 313 bp (mecA), 408 bp (PVL) and 516 bp (blaZ) of molecular weight. The standardization was performed in ATCC strains and Staphylococcus schleiferiand tested in 56 strains of Staphylococcusspp. The 16S gene (internal control) was amplified in all samples, mecA gene was detected in two samples, mecA associated with mecC gene in one sample, mecA associated to the blaZ gene in 14 samples and the blaZ gene in 15 samples. No resistance genes were amplified in 24 samples. The PVL gene was not amplified in any of the samples tested.

scholarly journals Proteomics characterization of Staphylococcus spp. from goat mastitis and phenogeno-typical assessment of resistance to beta-lactamics

2021 ◽  
Vol 41 ◽  
Author(s):  
Camila S. Pereira ◽  
Lídia M.M. Santos ◽  
Leandro S. Machado ◽  
Dayanne A. Melo ◽  
Shana M.O. Coelho ◽  
...  
Keyword(s):  
Clavulanic Acid ◽  
Meca Gene ◽  
Disk Diffusion ◽  
Penicillin G ◽  
Rrna Gene ◽  
Disk Diffusion Test ◽  
Maldi Tof ◽  
Two Samples ◽  
Amoxicillin Clavulanic Acid ◽  
Staphylococcus Spp

ABSTRACT: Mastitis occupies a prominent place among the diseases that affect dairy herds due to economic problems and public health. Staphylococcus spp. are infectious agents more involved in the etiology of caprine mastites, especially coagulase-negative Staphylococcus. Nineteen isolates of Staphylococcus spp. were obtained from subclinical caprine mastitis. All isolates were characterized by MALDI-TOF MS, being 47.36% (9/19) identified for S. epidermidis, 15.78% (3/19) for S. warneri, 10.52% (2/19) for S. aureus and S. caprae and 5.26% (1/19) for S. lugdunensis, S. simulans, and S. cohnii. All isolates characterized by MALDI-TOF were subjected a to polymerase chain reaction (PCR) for the 16S rRNA gene of Staphylococcus spp. to confirm the gender. After determining the species, tests for phenotypic detection of resistance to beta-lactams were carried out simple disk diffusion oxacillin, cefoxitin, penicillin G and amoxicillin + clavulanic acid, agar “screen” oxacillin and microdilution (MIC) cefoxitin. The disk diffusion test showed a strength of 58% (11/19) for penicillin G, 26.31% (5/19) for cefoxitin and 26.31% (5/19) for oxacillin. All strains were susceptible to amoxicillin + clavulanic acid and agar “screen” oxacillin. In the MIC, 63.15% (12/19) of the samples were cefoxitin resistant (MIC >4.0μg/ml). Then isolates were subjected to detection of the mecA resistance genes and regulators (mecl and mecRI), mecC and blaZ. Two samples of Staphylococcus epidermidis had the mecA gene. All isolates were negative for the mecA gene variant, mecl, mecRI, mecC and blaZ. These findings reinforce the importance of this group of microorganisms in the etiology of subclinical mastitis in goats and open perspectives for future research to investigate the epidemiology of the disease.

scholarly journals 643. Comparison of Multiplex Polymerase Chain Reaction (PCR) and Routine Culture for the Detection of Respiratory Pathogens in Pneumonia Patients

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S297-S297
Author(s):  
Emad Abu Sitta ◽  
Nicole Hubbard ◽  
Geehan Suleyman
Keyword(s):  
Resistance Genes ◽  
Standard Procedure ◽  
Meca Gene ◽  
Bacterial Pathogen ◽  
Antibiotic Resistance Genes ◽  
Antibiotic Use ◽  
Respiratory Pathogens ◽  
Culture Methods ◽  
Radiological Criteria ◽  
Two Samples

Abstract Background The identification of causative pathogens in pneumonia can be challenging, and conventional culture methods can take up to 72 hours. However, rapid microbiologic tests identify organisms within hours. The Biofire®Filmarray ((bioMérieux, North Carolina) Pneumonia Panel was recently approved by the FDA. The multiplex PCR system identifies 33 targets from sputum and bronchoalveolar (BAL) samples, which include 18 bacteria, 8 viruses, and 7 antibiotic resistance genes. The purpose is to compare the panel to routine culture methods for the detection of respiratory pathogens in patients with pneumonia in a 794-bed teaching hospital in northwest Ohio. Methods We retrospectively screened all hospitalized intensive care unit patients who met clinical and radiological criteria of pneumonia using electronic medical records between November 2018 and February 2019. Adult patients who had respiratory cultures collected within 7 days were included. Repeat specimens were excluded. Routine cultures were performed using the laboratory’s standard procedure, and Pneumonia Panel testing was performed according to manufacturer instructions. Results Fifty-nine respiratory or 13 BAL and 46 sputum specimens were evaluated. There was no discrepancy between culture and PCR in 63% (37/59) samples. One (8%) BAL and 10 (22%) sputum specimens had additional pathogens detected by PCR. There was a discrepancy between culture and PCR in four (31%) BAL and seven (15%) sputum samples. The largest discrepancy was noted amongst Serratia marcescens (4/59 or 7%) and Haemophilus influenzae (6/59 or 10%) species. Only one sputum culture had Legionella detected by PCR. Additionally, 17 specimens had a virus detected either alone or with another bacterial pathogen by PCR. For the resistance genes, KPC was detected by PCR but not by Modified Carbapenem Inactivation Method (mCIM) test. The mecA gene was detected in six of seven (86%) of methicillin-resistant Staphylococcus aureus (MRSA) isolates. CTX-M was detected in Serratia and Klebsiella pneunomiae in two samples; however, the organisms were not isolated in culture. Conclusion The Pneumonia Panel can identify additional bacteria that did not grow in culture. This panel can rapidly identify pathogens and potentially reduce unnecessary antibiotic use. Disclosures All authors: No reported disclosures

scholarly journals Rapid Detection of Equine Piroplasms Using Multiplex PCR and First Genetic Characterization of Theileria haneyi in Egypt

Pathogens ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 1414
Author(s):  
Bassma S. M. Elsawy ◽  
Ahmed M. Nassar ◽  
Heba F. Alzan ◽  
Raksha V. Bhoora ◽  
Sezayi Ozubek ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Genetic Characterization ◽  
Simultaneous Detection ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Theileria Equi ◽  
Babesia Caballi ◽  
Pcr Targeting ◽  
First Time

Equine Piroplasmosis (EP) is an infectious disease caused by the hemoprotozoan parasites Theileria equi, Babesia caballi, and the recently identified species T. haneyi. Hereby, we used a multiplex PCR (mPCR) targeting the 18S rRNA gene of T. equi and B. caballi for the simultaneous detection of EP in Egyptian equids and examined the presence of T. haneyi infections in Egypt. Blood samples from 155 equids (79 horses and 76 donkeys) collected from different governorates of Egypt were examined by mPCR and PCR targeting T. hayeni. The mPCR method revealed a prevalence of T. equi of 20.3% in horses and of 13.1% in donkeys and a prevalence of B. caballi of 1.2% in horses. B. caballi was not detected in donkeys in the current study. The mPCR method also detected coinfections with both species (2.5% and 1.3% in horses and donkeys, respectively). Additionally, we report the presence of T. haneyi in Egypt for the first time in 53.1% of the horse and 38.1% of the donkey tested samples. Coinfection with T. haneyi and T. equi was found in 13.5% of the samples, while infection with the three EP species was found in 1.9% of the samples.

scholarly journals Development of a simultaneous detection method of DNA markers linked to four disease and pest resistance genes in potato using multiplex PCR

2010 ◽  
Vol 12 (1) ◽  
pp. 22-25
Author(s):  
Kazuyuki Mori ◽  
Kengo Ohbayashi ◽  
Seiji Tamiya ◽  
Yu Sakamoto ◽  
Nobuhiro Mukojima ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Resistance Genes ◽  
Dna Markers ◽  
Detection Method ◽  
Simultaneous Detection ◽  
Pest Resistance

scholarly journals Multiplex PCR assay to detect rust resistance genes Lr26 and Lr37 in wheat

2010 ◽  
Vol 46 (No. 2) ◽  
pp. 85-89 ◽  
Author(s):  
T. Sumíková ◽  
A. Hanzalová
Keyword(s):  
Multiplex Pcr ◽  
Leaf Rust ◽  
Resistance Genes ◽  
Rust Resistance ◽  
Simultaneous Detection ◽  
Field Tests ◽  
Leaf Rust Resistance ◽  
Multiplex Pcr Assay ◽  
Winter Wheat Cultivars ◽  
Rust Resistance Genes

Multiplex PCR was developed and optimized for simultaneous detection of wheat leaf rust resistance genes Lr26 and Lr37. The presence of the genes was analyzed in 21 winter wheat cultivars registered in the Czech Republic. Gene Lr37 was detected in four tested cultivars (Bakfis, Biscay, Nicol, Mulan), gene Lr26 occurred only in one cultivar (Etela) and three cultivars (Clarus, Orlando and Rapsodia) were found to carry both these genes. Data obtained by PCR markers were compared with results of greenhouse and field tests. Seedling reactions of cultivars possessing Lr26 to seven different leaf rust isolates conformed to the results obtained by the marker analysis, however, there were found some discrepancies in the detections of Lr37, which could be detected in greenhouse seedling tests only with difficulties.

Retail Ready-to-Eat Food as a Potential Vehicle for Staphylococcus spp. Harboring Antibiotic Resistance Genes

2014 ◽  
Vol 77 (6) ◽  
pp. 993-998 ◽  
Author(s):  
WIOLETA CHAJĘCKA-WIERZCHOWSKA ◽  
ANNA ZADERNOWSKA ◽  
BEATA NALEPA ◽  
MAGDA SIERPI´NSKA ◽  
ŁUCJA ŁANIEWSKA-TROKENHEIM
Keyword(s):  
Antibiotic Resistance ◽  
Resistance Genes ◽  
Meca Gene ◽  
Antibiotic Resistance Genes ◽  
Tetracycline Resistance ◽  
Multidrug Resistant ◽  
Resistant Bacteria ◽  
Antibiotic Resistant ◽  
Cured Meats ◽  
Staphylococcus Spp

Ready-to-eat (RTE) food, which does not need thermal processing before consumption, could be a vehicle for the spread of antibiotic-resistant microorganisms. As part of general microbiological safety checks, staphylococci are routinely enumerated in these kinds of foods. However, the presence of antibiotic-resistant staphylococci in RTE food is not routinely investigated, and data are only available from a small number of studies. The present study evaluated the pheno- and genotypical antimicrobial resistance profile of Staphylococcus spp. isolated from 858 RTE foods (cheeses, cured meats, sausages, smoked fishes, salads). Of 113 strains isolated, S. aureus was the most prevalent species, followed by S. xylosus, S. saprophyticus, and S. epidermidis. More than half (54.9%) of the isolates were resistant to at least one class of tested antibiotic; of these, 35.4% of the strains were classified as multidrug resistant. Most of the isolates were resistant to cefoxitin (49.6%), followed by clindamycin (39.3%), tigecycline (27.4%), quinupristin-dalfopristin (22.2%), rifampin (20.5%), tetracycline (17.9%), and erythromycin (8.5%). All methicillin-resistant staphylococci harbored the mecA gene. Among the isolates resistant to at least one antibiotic, 38 harbored tetracycline resistance determinant tet(M), 24 harbored tet(L), and 9 harbored tet(K). Of the isolates positive for tet(M) genes, 34.2% were positive for the Tn916-Tn1545–like integrase family gene. Our results indicated that retail RTE food could be considered an important route for the transmission of antibiotic-resistant bacteria harboring multiple antibiotic resistance genes.

Development of a multiplex PCR method for simultaneous detection of diagnostic DNA markers of five disease and pest resistance genes in potato

Euphytica ◽  
2011 ◽  
Vol 180 (3) ◽  
pp. 347-355 ◽  
Author(s):  
Kazuyuki Mori ◽  
Yu Sakamoto ◽  
Nobuhiro Mukojima ◽  
Seiji Tamiya ◽  
Takashi Nakao ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Resistance Genes ◽  
Dna Markers ◽  
Simultaneous Detection ◽  
Pest Resistance ◽  
Pcr Method

Simultaneous Identification of Seven Foodborne Pathogens and Escherichia coli (Pathogenic and Nonpathogenic) Using Capillary Electrophoresis–Based Single-Strand Conformation Polymorphism Coupled with Multiplex PCR

2009 ◽  
Vol 72 (6) ◽  
pp. 1262-1266 ◽  
Author(s):  
MI-HWA OH ◽  
SE-HEE PAEK ◽  
GI WON SHIN ◽  
HAE-YEONG KIM ◽  
GYOO YEOL JUNG ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Capillary Electrophoresis ◽  
Multiplex Pcr ◽  
Simultaneous Detection ◽  
Pcr Amplification ◽  
Single Strand Conformation Polymorphism ◽  
Single Strand ◽  
Rrna Gene ◽  
Optimal Separation ◽  
Conformation Polymorphism

The objective of this study was to develop a novel technique for parallel analysis of eight important foodborne microbes using capillary electrophoresis–based single-strand conformation polymorphism (CE-SSCP) coupled with multiplex PCR. Specific primers for multiplex PCR amplification of the 16S rRNA gene were designed, corresponding to eight species of bacteria, including Escherichia coli, Clostridium perfringens, Campylobacter jejuni, Salmonella enterica, Listeria monocytogenes, Vibrio parahaemolyticus, Staphylococcus aureus, and Bacillus cereus, for the species-specific identification and optimal separation of their PCR products in subsequent analysis by CE-SSCP. Multiplex PCR conditions including annealing temperature, extension time, the number of PCR cycles, and primer concentrations were then optimized for simultaneous detection of all target foodborne bacteria. The diagnostic system using CE-SSCP combined with multiplex PCR developed here can be used for rapid investigation of causative agents of foodborne illness. The simplicity and high sensitivity of the method may lead to improved management of safety and illness related to food.

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