detection-and-isolation-of-campylobacter-spp-from-raw-meat

Detection and Isolation of <i>Campylobacter </i> spp. from Raw Meat 

This article presents a rapid yet robust protocol for isolating Campylobacter spp. from raw meats, specifically focusing on Campylobacter jejuni and ...

modified-most-probable-number-assay-to-quantify-salmonella-raw-ready

Modified Most Probable Number Assay to Quantify <em>Salmonella</em> in Raw and Ready-to-Cook Chicken Products

Salmonella is a leading cause of foodborne illness in the United States, particularly in poultry products. Traditional methods for detecting Salmonella ...

Agricultural Research Service (ARS), United States Department of Agriculture (USDA)

Using primers and fluorescent probes specific for the most common food-borne Campylobacter species (Campylobacter jejuni and Campylobacter coli), we developed a multiplex, most probable number (MPN) assay using quantitative PCR (qPCR) as the determinant for binomial detection: i.e., number of p positive pathogen growth responses out of n = 6 observations each of 4 mL (V) per dilution. Working with media washes of thrice frozen-thawed chicken pieces which had been spiked with known levels of C. jejuni and C. coli, we found that about 20% of the experiments had a significant amount of error in the form of either greater than 25% MPN calculation error (Δε) and/or a low apparent recovery rate (R less than 1 = MPN observed ÷ CFU spiked). Assuming such errors were exacerbated by an excessively small n, we examined computer-generated MPN enumeration data from the standpoint of stochastic sampling error (Δ) and found that such binomial-based assays behaved identically to Poisson-based methods (e.g., counting data) except that fewer technical replicates (n) appeared to be required for the same number of cells per test volume (μ). This result implies that the qPCR detection-based MPN protocol discussed herein should accurately enumerate a test population with a μ ≥ 1 using n = 6 observations per dilution. For our protocol, this equates to ≥ 8 cells per 400-500 g of sampled product. Based on this analysis, the error rate we saw in spiked experiments (where μ >> 1) implied a non-stochastic source. In other experiments we present evidence that this source was, at least in part, related to the cell concentration step (i.e., centrifugation). We also demonstrate that the error rate lessened (from ~38% to ~13%) at lower Campylobacter levels (μ ≤ 40) as would most likely exist in nature. Using this protocol, we were able to quantify 14 to 1,226 MPN per 450 g of naturally contaminated chicken for skinless pieces and 11 to 244 MPN per 450 g for wings, breasts, legs, and thighs (skin on) whereupon about 50% of the 29 samples tested negative for both species. Four of these chicken wash samples did have substantially lower Campylobacter levels (1 to 6 MPN per 450 g) which might be better enumerated using a larger n. However, we established that the limit of quantification of this protocol diminishes for n > 6 because one is ever more diluting the sample, or lessening V, to achieve the requisite n.

Non-stochastic sampling error in quantal analyses for Campylobacter species on poultry products.

Shiga toxins 1 and 2 (Stx1 and Stx2) from Shiga toxin-producing E. coli (STEC) bacteria were simultaneously detected with a newly developed, high-throughput antibody microarray platform. The proteinaceous toxins were immobilized and sandwiched between biorecognition elements (monoclonal antibodies) and pooled horseradish peroxidase (HRP)-conjugated monoclonal antibodies. Following the reaction of HRP with the precipitating chromogenic substrate (metal enhanced 3,3-diaminobenzidine tetrahydrochloride or DAB), the formation of a colored product was quantitatively measured with an inexpensive flatbed page scanner. The colorimetric ELISA microarray was demonstrated to detect Stx1 and Stx2 at levels as low as ~4.5 ng/mL within ~2 h of total assay time with a narrow linear dynamic range of ~1-2 orders of magnitude and saturation levels well above background. Stx1 and/or Stx2 produced by various strains of STEC were also detected following the treatment of cultured cells with mitomycin C (a toxin-inducing antibiotic) and/or B-PER (a cell-disrupting, protein extraction reagent). Semi-quantitative detection of Shiga toxins was demonstrated to be sporadic among various STEC strains following incubation with mitomycin C; however, further reaction with B-PER generally resulted in the detection of or increased detection of Stx1, relative to Stx2, produced by STECs inoculated into either axenic broth culture or culture broth containing ground beef.

A high-throughput, precipitating colorimetric sandwich ELISA microarray for Shiga toxins.

Antibody microarray is a powerful analytical technique because of its inherent ability to simultaneously discriminate and measure numerous analytes, therefore making the technique conducive to both the multiplexed detection and identification of bacterial analytes (i.e., whole cells, as well as associated metabolites and/or toxins). We developed a sandwich fluorescent immunoassay combined with a high-throughput, multiwell plate microarray detection format. Inexpensive polystyrene plates were employed containing passively adsorbed, array-printed capture antibodies. During sample reaction, centrifugation was the only strategy found to significantly improve capture, and hence detection, of bacteria (pathogenic Escherichia coli O157:H7) to planar capture surfaces containing printed antibodies. Whereas several other sample incubation techniques (e.g., static vs. agitation) had minimal effect. Immobilized bacteria were labeled with a red-orange-fluorescent dye (Alexa Fluor 555) conjugated antibody to allow for quantitative detection of the captured bacteria with a laser scanner. Shiga toxin 1 (Stx1) could be simultaneously detected along with the cells, but none of the agitation techniques employed during incubation improved detection of the relatively small biomolecule. Under optimal conditions, the assay had demonstrated limits of detection of ~5.8 × 10⁵ cells/mL and 110 ng/mL for E. coli O157:H7 and Stx1, respectively, in a ~75 min total assay time.

Antibody Microarray for E. coli O157:H7 and Shiga Toxin in Microtiter Plates.

Magnesium oxide nanoparticles (MgO nanoparticles, with average size of 20 nm) have considerable potential as antimicrobial agents in food safety applications due to their structure, surface properties, and stability. The aim of this work was to investigate the antibacterial effects and mechanism of action of MgO nanoparticles against several important foodborne pathogens.

Study on the mechanism of antibacterial action of magnesium oxide nanoparticles against foodborne pathogens.

The complete genome sequence data of  SJTUF_J27 isolated from seaweed in China is reported here. The size of the genome is 2.8 Mbp with 32.9% G + C content, consisting of 2614 coding sequences and 77 RNAs. A number of virulence factors, including antimicrobial resistance genes (fluoroquinolone, beta-lactams, fosfomycin, mupirocin, trimethoprim, and aminocoumarin) and the  enterotoxin cluster, were found in the genome. In addition, the genes encoding metal-binding proteins and associated heavy metal resistance were identified. Phylogenetic data analysis, based upon genome-wide single nucleotide polymorphisms (SNPs), and comparative genomic evaluation with BLAST Ring Image Generator (BRIG) were performed for SJTUF_J27 and four  strains isolated from food. The completed genome data was deposited in NCBI׳s GenBank under the accession number CP019117, https://www.ncbi.nlm.nih.gov/nuccore/CP019117.

Whole-genome sequence data and analysis of a  strain SJTUF_J27 isolated from seaweed.

Pulsed-field gel electrophoresis (PFGE) is one of the most effective genotyping methods for the study of genetic relatedness of microorganisms as well as for epidemiologic investigation of outbreaks caused by pathogenic bacteria, including Staphylococcus aureus. The technique relies on the size patterns of large DNA fragments generated by the cleavage of intact bacterial chromosomes with a rare cutting restriction enzyme, and subsequently resolved by pulsed-field electrophoresis with periodic changes of the orientation of the electrical field across the gel. The high discriminatory power, improved reproducibility by standardization of experimental protocols and data analysis guidelines, and establishment of a national PFGE database of S. aureus profiles have made it a valuable means for global tracking of S. aureus infection sources and determination of genetic relatedness of the strains.

Pulsed-Field Gel Electrophoresis Typing of Staphylococcus aureus Strains.

The complete genome sequence of  YH003, isolated from retail chicken, was determined using PacBio and Illumina technologies. The assembled genome is 1,743,985 bp (G+C content of 30.3%). Genome annotation revealed several genes encoding virulence and antibiotic resistance factors, including a type VI secretion system, cytolethal distending toxins, and a multidrug efflux system.

Complete Genome Sequence and Annotation of Campylobacter jejuni YH003, Isolated from Retail Chicken.

Raw poultry can harbor microbial pathogens. Campylobacter jejuni BSD5, isolated from a critical control point within a poultry production plant, was sequenced. Genome annotation revealed several virulence genes including antibiotic resistance genes in agreement with the phenotypic results, indicating a potential risk of this strain to public health.

Complete Genome Sequence of Campylobacter jejuni BSD5, a Multidrug-Resistant Isolate from a Poultry Processing Facility in the United States.

 is a leading bacterial cause of gastrointestinal infections in humans and has imposed substantial medical and public health burdens worldwide. Among a total of 39 species in the  genus,  is the most important species responsible for approx. 90% of human  illness. Most cases of the infection were acquired by ingesting undercooked poultry meat due to the high prevalence of  in the products. Here, we reported the dataset of raw sequences, de novo assembled and annotated genomes of  strains S27, S33, and S36 recently isolated from retail chicken by using PacBio highly accurate long-read sequencing technology combined with bioinformatics tools. Our data revealed several virulence and antibiotic resistance genes in each of the chromosomes, a type IV secretion system in the plasmid (pCjS33) of  S33, and a type VI secretion system and a phage in the plasmid (pCjS36) of  S36. This study not only provides new sequence data but also extends the knowledge pertaining to the genomic and functional aspects of this important foodborne pathogen, including the genetic determinants of virulence and antibiotic resistance.

Whole Genome Sequences, De Novo Assembly, and Annotation of Antibiotic Resistant  Strains S27, S33, and S36 Newly Isolated from Chicken Meat.

 is one of the leading bacterial causes of gastroenteritis worldwide. It frequently contaminates poultry and other raw meat products, which are the primary sources of  infections in humans. Plasmids, known as important mobile genetic elements, often carry genes for antibiotic resistance, virulence, and self-mobilization. They serve as the main vectors for transferring genetic material and spreading resistance and virulence among bacteria. In this study, we identified 34 new plasmids from 43  and  strains isolated from retail meat using long-read and short-read genome sequencing. Pangenomic analysis of the plasmid assemblies and reference plasmids from GenBank revealed five distinct groups, namely, pTet, pVir, mega plasmids (>80 kb), mid plasmids (~30 kb), and small plasmids (<6 kb). Pangenomic analysis identified the core and accessory genes in each group, indicating a high degree of genetic similarity within groups and substantial diversity between the groups. The pTet plasmids were linked to tetracycline resistance phenotypes in host strains. The mega plasmids carry multiple genes (e.g., (3')-III, type IV and VI secretion systems, and type II toxin-antitoxin systems) important for plasmid mobilization, virulence, antibiotic resistance, and the persistence of . Together, the identification and comprehensive genetic characterization of new plasmids from  food isolates contributes to understanding the mechanisms of gene transfer, particularly the spread of genetic determinants of virulence and antibiotic resistance in this important pathogen.

Yiping He

Agricultural Research Service (ARS), United States Department of Agriculture (USDA)

Detection and Isolation of Campylobacter spp. from Raw Meat

Modified Most Probable Number Assay to Quantify Salmonella in Raw and Ready-to-Cook Chicken Products