ABSTRACT
The increasing consumption of organic or ready-to-eat food may cause serious foodborne disease outbreaks. Developing microbiological culture for detection of food-borne pathogens is time-consuming, expensive, and laborious. Thus, alternative methods such as polymerase chain reaction (PCR) are usually employed for outbreaks investigation. In this work, we aimed to develop a rapid and simple protocol for the simultaneous detection of Escherichia coli (E coli), Listeria monocytogenes (L. monocytogenes), Staphylococcus aureus (S. aureus) and Salmonella enterica (S. enterica), by the combination of an enrichment step in a single culture broth and a multiplex PCR (mPCR) assay. The effectiveness of several enrichment media was assessed by culture and PCR. Buffered peptone water (BPW) was selected as the optimum one. Then, mPCR conditions were optimized and applied both to pure co-cultures and artificially inoculated food samples (organic lettuce and minced meat). In the culture medium inoculated at 100 CFU/mL, mPCR was able to detect the four microorganisms. When performed on artificially food samples, the mPCR assy was able to detect E. coli, S. enterica, and L. monocytogenes. In conclusion, BPW broth can effectively support the simultaneous growth of E. coli, S. aureus, L. monocytogenes, and S. enterica and could be, thus, used prior to a mPCR detection assay in ready-to-eat food, thereby considerably reducing the time, efforts and costs of analyzes.
Subject(s)
Escherichia coli O157 , Listeria monocytogenes , Food Microbiology , Listeria monocytogenes/genetics , Multiplex Polymerase Chain Reaction/methods , Staphylococcus aureus/geneticsABSTRACT
The aim of this work was to assess the effect of chlorine water treatment on Arcobacter butzleri and to study the survival strategies of this organism in chlorinated and non-chlorinated drinking water. A. butzleri NCTC 12481 was inoculated into chlorinated and non-chlorinated water and samples were removed aseptically, immediately and periodically during the next 2 days (for chlorinated drinking water) or 35 days (for non-chlorinated drinking water). The membrane integrity (Live/Dead BacLight kit), 16S rRNA (FISH technique), DNA content (23S rRNA PCR-RFLPs) and culturability changes in A. butzleri cells were analyzed. Culturability of the cells was lost at 5 min in chlorinated drinking water. At that time the cells showed membrane damage, although fluorescent intensity of 16S rRNA hybridization was constant throughout the chlorine treatment. After 48 h the amplicon specific for the 23S rRNA gene was weakly detected. In non-chlorinated drinking water cells lost their culturability after 16 days but the other factors measured indicated that Arcobacter remained viable throughout the experiment.
Subject(s)
Arcobacter/drug effects , Arcobacter/growth & development , Chlorine/pharmacology , Fresh Water/microbiology , Water Supply , Arcobacter/genetics , Cell Membrane Permeability/drug effects , Colony Count, Microbial , DNA, Ribosomal/analysis , Disinfection/methods , In Situ Hybridization, Fluorescence , Microscopy, Electron, Scanning , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , RNA, Ribosomal, 23S/geneticsABSTRACT
The aim of this study was to evaluate PCR and fluorescent in situ hybridization (FISH) techniques for detecting Arcobacter and Campylobacter strains in river water and wastewater samples. Both 16S and 23S rRNA sequence data were used to design specific primers and oligonucleotide probes for PCR and FISH analyses, respectively. In order to assess the suitability of the methods, the assays were performed on naturally and artificially contaminated samples and compared with the isolation of cells on selective media. The detection range of PCR and FISH assays varied between 1 cell/ml (after enrichment) to 10(3) cells/ml (without enrichment). According to our results, both rRNA-based techniques have the potential to be used as quick and sensitive methods for detection of campylobacters in environmental samples.
