ABSTRACT
A single-tube multiplex real-time PCR targeting the nuclease (nuc) gene and subsequent high-resolution melting analysis (HRMA) were used to identify 13 different Staphylococcus species. The nuc gene was targeted due to its low intraspecies variation and the greater interspecies variation than the 16S rRNA gene in Staphylococcus. We used HRMA software that can store and compare HRMA profiles from different runs as long as the runs contain the same reference reaction. Thus, we reduced the 14 PCRs to 2 different PCRs, one targeting the unknown sequence and the other targeting the reference sequences to screen 13 different Staphylococcus species. The specificity of the developed method was tested on 16 different Staphylococcus reference strains and 115 different field strains that were isolated from the milk of cattle with subclinical mastitis. We conclude that the method can be used to quickly and cost-effectively differentiate Staphylococcus aureus (S. aureus) from other Staphylococcus species (S. epidermidis, S. lugdunensis, S. schleiferi, S. hyicus, S. chromogenes, S. lentus, S. haemolyticus, S. xylosus, S. saprophyticus, S. warneri, S. simulans and S. hominis).
Subject(s)
Polymerase Chain Reaction/methods , Staphylococcus/genetics , Staphylococcus/isolation & purification , Animals , Bacterial Proteins/genetics , Cattle , DNA, Bacterial/analysis , DNA, Bacterial/genetics , Female , Mastitis/diagnosis , Mastitis/microbiology , Micrococcal Nuclease/genetics , Milk/microbiology , RNA, Ribosomal, 16S/genetics , Software , Species Specificity , Staphylococcal Infections/diagnosis , Staphylococcal Infections/microbiology , Staphylococcus/classificationABSTRACT
This study examined the incidence of Clostridium perfringens in raw, ready-to-cook (RTC), and ready-to-eat (RTE) meat and meat-based products (N = 306) collected from restaurants, supermarkets, and butcher shops in Bursa, Turkey. In addition, we investigated the presence of the C. perfringens enterotoxin (CPE), as well as cpe genes and their source (chromosomal or plasmid borne). In this study, tryptose sulfite cycloserine (TSC) agar for classic culture isolation and API and real-time polymerase chain reaction (RT-PCR) techniques were used to identify C. perfringens and detect cpa and cpe genes from these products, respectively. Seventeen C. perfringens isolates (5.6%) were isolated and identified with API 20A. In addition, 42 of 81 suspicious isolates (51.9%) were identified as C. perfringens using RT-PCR. Of the 81 suspicious isolates tested by RT-PCR, 22 (27.2%) carried the cpe gene either on the plasmid or chromosome. Twenty-one isolates were positive for chromosomal cpe (C-cpe), and one was positive for plasmid-borne cpe (P-cpe). CPE was detected in 31.8% (7/22) of the cpe positive isolates by the PET-RPLA test. In conclusion, C. perfringens and their CPEs were present in raw, RTC, and RTE meat and meat-based foods in this study. It is emphasized that the presence of C. perfringens and the cpe gene in these foods may be a potential risk for human health.
Subject(s)
Clostridium perfringens/isolation & purification , Food Contamination/analysis , Food Microbiology , Meat Products/microbiology , Meat/microbiology , Bacterial Proteins/genetics , Enterotoxins/isolation & purification , Plasmids , Polymerase Chain Reaction , TurkeyABSTRACT
BACKGROUND: Escherichia coli is one of the major causative agents of bovine mastitis worldwide, and is typically associated with acute, clinical mastitis. Besides this, E. coli strains which belong to the extra-intestinal pathogenic group are also the major cause of urinary tract infections and pyometra in dogs. OBJECTIVES: In this study, it was aimed to investigate phylo-groups/subgroups in 155 E. coli isolates obtained from acute bovine mastitis, 43 from urinary tract infections of dogs and 20 from canine pyometra by a formerly described triplex PCR and recently described new quadruplex polymerase chain reaction (PCR) method. RESULTS: Group A1 (n = 118; 76%) and B1 (n = 71; 46%) were found to be the most prevalent groups by triplex and quadruplex PCR assays in mastitis isolates, respectively. Phylo-typing of 43 urinary tract isolates also revealed that most of the isolates belonged to A1 (n = 23; 54%) by triplex and B2 (n = 36; 84%) by quadruplex PCR assays. The isolates assigned as group A1 (n = 17; 85%) by triplex PCR could not be classified by quadruplex PCR in pyometra isolates. CONCLUSIONS: The results support the hypothesis that E. coli strains isolated from bovine mastitis cases are environmental. Also, groups C, E and F were identified as new phylo-groups for the first time in acute bovine mastitis cases. The comparison of triplex PCR with quadruplex PCR results revealed that most of the groups assigned in triplex PCR were altered by quadruplex PCR assay.
