Category Archives: PCR

Research – Occurrence, Antibiotic Resistance and Biofilm-Forming Ability of Listeria monocytogenes in Chicken Carcasses and Cuts

Posted on December 6, 2024 by | Leave a comment

MDPI

A total of 104 samples of chicken meat acquired on the day of slaughter from two slaughterhouses in northwestern Spain were analyzed. These comprised 26 carcasses and 26 cuts from each of the two establishments. An average load of 5.39 ± 0.61 log10 cfu/g (total aerobic counts) and 4.90 ± 0.40 log10 cfu/g (psychrotrophic microorganisms) were obtained, with differences (p < 0.05) between types of samples and between slaughterhouses. Culturing methods involving isolation based on the UNE-EN-ISO 11290-1:2018 norm and identification of isolates by polymerase chain reaction (PCR) to detect the lmo1030 gene allowed the detection of Listeria monocytogenes in 75 samples (72.1% of the total; 50.0% of the carcasses and 94.2% of the cuts). The 75 isolates, one for each positive sample, were tested for resistance against a panel of 15 antibiotics of clinical interest by the disc diffusion method. All isolates belonged to the serogroup IIa (multiplex PCR assay) and showed resistance to between four and ten antibiotics, with an average value of 5.7 ± 2.0 resistances per isolate, this rising to 7.0 ± 2.1 when strains with resistance and reduced susceptibility were taken together. A high prevalence of resistance was observed for antibiotics belonging to the cephalosporin and quinolone families. However, the level of resistance was low for antibiotics commonly used to treat listeriosis (e.g., ampicillin or gentamicin). Nine different resistance patterns were noted. One isolate with each resistance pattern was tested for its ability to form biofilms on polystyrene during 72 h at 12 °C. The total biovolume of the biofilms registered through confocal laser scanning microscopy (CLSM) in the observation field of 16,078.24 μm2 ranged between 13,967.7 ± 9065.0 μm3 and 33,478.0 ± 23,874.1 μm3, and the biovolume of inactivated bacteria between 0.5 ± 0.4 μm3 and 179.1 ± 327.6 μm3. A direct relationship between the level of resistance to antibiotics and the ability of L. monocytogenes strains to form biofilms is suggested.

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Posted in Antibiotic Resistance, antimicrobial resistance, Biofilm, Food Microbiology Research, Listeria, Listeria monocytogenes, PCR, Research

Research – Verification of a Rapid Analytical Method for the Qualitative Detection of Listeria spp. and Listeria monocytogenes by a Real-Time PCR Assay according to EN UNI ISO 16140-3:2021

Posted on February 9, 2024 by | Leave a comment

MDPI

Abstract

Microbial contamination and foodborne infections are a significant global public health concern. For this reason, the detection, monitoring, and characterization of pathogens represent a significant challenge in quality control settings. Standard approaches, such as culture methods and biochemical tests, are known to be very time-consuming and intensive. Conversely, molecular technologies based on the genomic identification of bacteria are quick and low-cost. Listeria monocytogenes is an opportunistic pathogen and a major concern especially in food industries. It is important to understand and implement multiple quality control measures to control Listeria infection risk and prevent the contamination of products. Standardized detection and confirmation tests such as the API Listeria test, MALDI-TOF MS, and PCR analysis are available. The aim of our work is to provide a specific molecular method, designed according to the EN UNI ISO 16140-3:2021, for the specific detection, monitoring, and characterization of Listeria spp. and Listeria monocytogenes contamination. The verification of this new rapid approach by real-time PCR (qPCR) overcomes the limitations of culture-based techniques, meeting all the verification criteria required by ISO guidelines, including implementation and item confirmation. This system offers a powerful approach to the real-time assessment of food safety, useful for industry self-monitoring and regulatory inspection.

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Posted in Food Microbiology Research, ISO Standards, Listeria, Listeria monocytogenes, PCR, Research

Research – Shiga toxin-producing Escherichia coli (STEC) in meat and leafy greens available in the Swedish retail market – Occurrence and diversity of stx subtypes and serotypes

Posted on January 23, 2024 by | Leave a comment

Science Direct

Abstract

Shiga toxin-producing Escherichia coli (STEC) is a major cause of foodborne illness, ranging from mild diarrhea to permanent kidney failure. This study summarizes the results of four surveys performed at different time periods, which investigated the occurrence and characteristics of STEC in beef, lamb and leafy greens available in the Swedish retail market. Such data is required when assessing the public health risk of varying types of STEC in different foods, and for establishing risk management measures. Samples from domestic and imported products were collected based on their availability in the retail market. The occurrence of STEC was investigated in 477 samples of beef, 330 samples of lamb and 630 samples of leafy greens. The detection of virulence genes (stx1, stx2, eae) was performed using real-time PCR followed by the isolation of bacteria from stx-positive enriched samples using immunomagnetic separation or an immunoblotting method. All STEC isolated from the food samples was further characterised in terms of stx subtyping and serotyping through whole genome sequencing. STEC was isolated from 2 to 14 % of beef samples and 20 to 61 % of lamb samples, depending on the region of origin. STEC was not isolated from samples of leafy greens, although stx genes were detected in 11 (2 %) of the samples tested. In total, 5 of the 151 sequenced STEC isolates from meat contained stx2 and eae, of which 4 such combinations had the stx2a subtype. The stx2 gene, stx2a in particular, is strongly associated with serious disease in humans, especially in combination with the eae gene. The isolates belonged to 20 different serotypes. Two isolates from beef and one from lamb belonged to the serotype O157:H7 and contained genes for stx2 and eae. Overall, several combinations of stx subtypes were found in isolates from beef, whereas stx1c, either alone or together with stx2b, was the dominant combination found in STEC from lamb. In conclusion, STEC was rare in whole meat samples of domestic beef in the Swedish retail market, whereas such bacteria were frequently found in minced meat and whole meat samples of imported beef and domestic and imported lamb. Although the number of isolates containing genes linked to an increased risk of severe disease was low, beef and lamb in the Swedish retail market is a common source of human exposure to potentially pathogenic STEC.

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Posted in eae, eaeA, Food Microbiology Research, PCR, Research, Shigatoxin, STEC, STEC E.coli, STX 1, STX 2

Research – Isolation of Listeria monocytogenes from poultry red mite (Dermanyssus gallinae) infesting a backyard chicken farm in Greece

Posted on January 22, 2023 by | Leave a comment

Nature.com

The poultry red mite (PRM), Dermanyssus gallinae, is arguably the most harmful, ubiquitous haematophagous ectoparasite infesting egg-laying hens. PRM is a vector of various microorganisms, with some being important for food microbiology and public health. The present study aimed to investigate the presence of specific pathogens, including Escherichia coli, Salmonella spp. and Listeria spp., carried by PRM infesting a chicken farm in Greece. Mites were caught using cardboard traps (Avivet), and 100 unwashed PRM were homogenized and used for microbiological cultures. Microbiological cultures were carried out on general and selective substrates to detect the above-mentioned bacteria. Specifically for Listeria spp., DNA was extracted from bacteria grown in Tryptone Soya Yeast Extract Agar using a commercial kit. The hly gene encoding the Listeriolysin O protein was amplified by PCR. Mites were identified as D. gallinae using morphological keys as well as by COI DNA barcoding. Microbiological cultures and PCR assays were positive for Listeria monocytogenes. No other bacteria were detected. The current study constitutes the first molecular isolation of L. monocytogenes from D. gallinae, confirming that PRM can carry this food-borne pathogen. PRM control measures and hygiene practices should be applied to minimize any possible contamination risk of poultry products with L. monocytogenes and safeguard public health.

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Posted in DNA, E.coli, Food Microbiology Research, Listeria, Listeria monocytogenes, Microorganism DNA, PCR, Poultry Red Mite, Research, Salmonella

Research – Evaluation of PCR Detection of Salmonella in Alfalfa Sprouts

Posted on February 18, 2013 by | Leave a comment

Ingenta ConnectEurofins

Abstract:

This study evaluated the efficacy of a PCR-based system (DuPont Qualicon BAX) for detection of Salmonella in sprouts and spent irrigation water collected during sprouting of seeds naturally contaminated with Salmonella. Alfalfa seeds were grown in Mason jars at 20 and 30°C for 3 days. Levels of Salmonella present in the water and sprouts were determined by most-probable-number (MPN) analysis. Background microflora levels were also determined. Samples of spent irrigation water and sprouts were enriched overnight individually in tetrathionate broth and in buffered peptone water with novobiocin at 42°C and then run in the BAX system. Samples were also enriched according to the U.S. Food and Drug Administration’s Bacteriological Analytical Manual (FDA BAM) method for Salmonella as a comparison. Salmonella levels were lower at 20°C compared with 30°C for some trials, and background microflora levels ranged from 107 to 108 CFU/g or ml at 20°C and 108 to 109 CFU/g or ml at 30°C. In trials with a Salmonella level >1.1 MPN/g or ml, both the BAX and FDA BAM methods were able to detect Salmonella in all samples. In trials with lower levels (0.21 MPN/g or ml or lower) of Salmonella, BAX was able to detect more positive samples than FDA BAM. For one trial with <0.003 MPN/g or ml of Salmonella, the presence of the pathogen was not indicated by either the BAX or the FDA BAM method. The results suggest that PCR detected low levels of Salmonella in sprouts or spent irrigation water collected from sprouting of naturally contaminated seeds.

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Posted in Bacteria, Food Hygiene, Food Inspections, Food Microbiology, Food Safety, Food Technology, Food Testing, Methods, Microbiology, PCR, Salmonella

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