Detection of Extended-Spectrum β-Lactamase (ESBL) E. coli at Different Processing Stages in Three Broiler Abattoirs

The European Food Safety Authority (EFSA) identified extended-spectrum β-lactamase/AmpC β-lactamase (ESBL/AmpC)-producing E. coli as one of the main priority hazards for poultry. Different studies detected ESBL-producing E. coli at broiler fattening farms and in abattoirs, concluding that poultry meat is a potential source of human infection. Broiler breast skin samples taken in three abattoirs with different scalding techniques were examined for ESBL-producing Escherichia (E.) coli and their phylogenetic groups. A total of 307 ESBL-producing E. coli isolates were found, and the abattoir with conventional immersion scalding with thermal treatment of the water had the lowest incidence. Phylogroups D/E and B1 were mostly detected, while phylogroups C, D, and E were not detected. Phylogroup B2 was detected in low proportions. The phylogroups B2 and D are important as they have been associated with urinary tract infections in humans, but were only detected in low proportions at different processing stages in this study. Since the risk for the consumer of being infected via chicken meat with ESBL-producing E. coli and E. coli of highly pathogenic phylogroups cannot be excluded, good kitchen hygiene is of great importance

Closely related Campylobacter jejuni strains from different sources reveal a generalist rather than a specialist lifestyle

Background: Campylobacter jejuni and Campylobacter coli are human intestinal pathogens of global importance. Zoonotic transmission from livestock animals or animal-derived food is the likely cause for most of these infections. However, little is known about their general and host-specific mechanisms of colonization, or virulence and pathogenicity factors. In certain hosts, Campylobacter species colonize persistently and do not cause disease, while they cause acute intestinal disease in humans. Results: Here, we investigate putative host-specificity using phenotypic characterization and genome-wide analysis of genetically closely related C. jejuni strains from different sources. A collection of 473 fresh Campylobacter isolates from Germany was assembled between 2006 and 2010 and characterized using MLST. A subset of closely related C. jejuni strains of the highly prevalent sequence type ST-21 was selected from different hosts and isolation sources. PCR typing of strain- variable genes provided evidence that some genes differed between these strains. Furthermore, phenotypic variation of these strains was tested using the following criteria: metabolic variation, protein expression patterns, and eukaryotic cell interaction. The results demonstrated remarkable phenotypic diversity within the ST-21 group, which however did not correlate with isolation source. Whole genome sequencing was performed for five ST-21 strains from chicken, human, bovine, and food sources, in order to gain insight into ST-21 genome diversity. The comparisons showed extensive genomic diversity, primarily due to recombination and gain of phage-related genes. By contrast, no genomic features associated with isolation source or host were identified. Conclusions: The genome information and phenotypic data obtained in vitro and in a chicken infection model provided little evidence of fixed adaptation to a specific host. Instead, the dominant C. jejuni ST-21 appeared to be characterized by phenotypic flexibility and high genetic microdiversity, revealing properties of a generalist. High genetic flexibility might allow generalist variants of C. jejuni to reversibly express diverse fitness factors in changing environments

Closely related Campylobacter jejuni strains from different sources reveal a generalist rather than a specialist lifestyle

Background: Campylobacter jejuni and Campylobacter coli are human intestinal pathogens of global importance. Zoonotic transmission from livestock animals or animal-derived food is the likely cause for most of these infections. However, little is known about their general and host-specific mechanisms of colonization, or virulence and pathogenicity factors. In certain hosts, Campylobacter species colonize persistently and do not cause disease, while they cause acute intestinal disease in humans. Results: Here, we investigate putative host-specificity using phenotypic characterization and genome-wide analysis of genetically closely related C. jejuni strains from different sources. A collection of 473 fresh Campylobacter isolates from Germany was assembled between 2006 and 2010 and characterized using MLST. A subset of closely related C. jejuni strains of the highly prevalent sequence type ST-21 was selected from different hosts and isolation sources. PCR typing of strain-variable genes provided evidence that some genes differed between these strains. Furthermore, phenotypic variation of these strains was tested using the following criteria: metabolic variation, protein expression patterns, and eukaryotic cell interaction. The results demonstrated remarkable phenotypic diversity within the ST-21 group, which however did not correlate with isolation source. Whole genome sequencing was performed for five ST-21 strains from chicken, human, bovine, and food sources, in order to gain insight into ST-21 genome diversity. The comparisons showed extensive genomic diversity, primarily due to recombination and gain of phage-related genes. By contrast, no genomic features associated with isolation source or host were identified. Conclusions: The genome information and phenotypic data obtained in vitro and in a chicken infection model provided little evidence of fixed adaptation to a specific host. Instead, the dominant C. jejuni ST-21 appeared to be characterized by phenotypic flexibility and high genetic microdiversity, revealing properties of a generalist. High genetic flexibility might allow generalist variants of C. jejuni to reversibly express diverse fitness factors in changing environments

Serotype Distribution of Salmonella Isolates from Turkey Ground Meat and Meat Parts

The aim of the study was to find out the serotype distribution of 169 Salmonella colonies recovered from 112 Salmonella positive ground turkey (115 colonies) and 52 turkey meat parts (54 colonies). Out of 15 Salmonella serotypes: S. Corvallis, S. Kentucky, S. Bredeney, S. Virchow, S. Saintpaul and S. Agona were identified as the predominant serovars at the rates of 27%, 13%, 12%, 12%, 11%, and 10%, respectively. Other serotypes were below 6% of the total isolates. All S. Kentucky and S. Virchow and most of the S. Corvallis (39/46) and S. Heidelberg (9/9) serotypes were recovered from ground turkey. The results indicate that turkey ground meat and meat parts were contaminated with quite distinct Salmonella serotypes. This is the first study reporting Salmonella serotype distribution in turkey meat and S. Corvallis as predominant serotype in poultry meat in Turkey

"Limits of control"--crucial parameters for a reliable quantification of viable campylobacter by real-time PCR.

The unsuitability of the "CFU" parameter and the usefulness of cultivation-independent quantification of Campylobacter on chicken products, reflecting the actual risk for infection, is increasingly becoming obvious. Recently, real-time PCR methods in combination with the use of DNA intercalators, which block DNA amplification from dead bacteria, have seen wide application. However, much confusion exists in the correct interpretation of such assays. Campylobacter is confronted by oxidative and cold stress outside the intestine. Hence, damage caused by oxidative stress probably represents the most frequent natural death of Campylobacter on food products. Treatment of Campylobacter with peroxide led to complete loss of CFU and to significant entry of any tested DNA intercalator, indicating disruption of membrane integrity. When we transiently altered the metabolic state of Campylobacter by abolishing the proton-motive force or by inhibiting active efflux, CFU was constant but enhanced entry of ethidium bromide (EtBr) was observed. Consistently, ethidium monoazide (EMA) also entered viable Campylobacter, in particular when nutrients for bacterial energization were lacking (in PBS) or when the cells were less metabolically active (in stationary phase). In contrast, propidium iodide (PI) and propidium monoazide (PMA) were excluded from viable bacterial cells, irrespective of their metabolic state. As expected for a diffusion-limited process, the extent of signal reduction from dead cells depended on the temperature, incubation time and concentration of the dyes during staining, prior to crosslinking. Consistently, free protein and/or DNA present in varying amounts in the heterogeneous matrix lowered the concentration of the DNA dyes at the bacterial membrane and led to considerable variation of the residual signal from dead cells. In conclusion, we propose an improved approach, taking into account principles of method variability and recommend the implementation of process sample controls for reliable quantification of intact and potentially infectious units (IPIU) of Campylobacter by real-time PCR

Loss of DNA upon cell inactivation.

<p>Data stem from at least seven independent experiments using bacterial suspensions from all growth phases.</p

Fluorimetric analysis of EtBr and PI entry into <i>C. jejuni</i> DSM 4688 at different metabolic states and during oxidative stress.

<p>Cells were grown to either exponential phase (OD 0.3–0.7) or stationary phase (OD 1.5–1.7) and diluted to OD 0.2 for the fluorimetric assay. At the timepoint  = 0, <i>C. jejuni</i> were confronted with either 100 µM EtBr (upper panel) or 100 µM PI (lower panel) and with distinct effectors. Black, control without effector; red, 150 µg/ml of the efflux inhibitor PAβN; green, 100 µM of the protonophor CCCP; blue, 5% H₂O₂, leading to <i>C. jejuni</i> cell death. While EtBr enters <i>C. jejuni</i> depending on the metabolic state, PI is passively excluded from viable cells. Mean fluorescence intensities (in 100% of the maximal fluorescence of the respective dead (H₂O₂) cell suspension reached after saturation (for stationary phase in the presence of PI 100% value was reached after 40 min)) and standard deviations are depicted from at least three independent experiments.</p

Schematic view of the quantification approach.

<p>The sample is aliquoted to four tubes, two of which (tube 2 and 4) are spiked with the sample process control (SPC), a distinct number of dead <i>C. jejuni</i> (e. g. 10⁷ H₂O₂-treated <i>C. jejuni</i>). In order to lower the amount of free DNA and/or protein, potentially interfering with the dose-effect relation of PMA, the samples are centrifuged and resuspended into PBS. After PMA addition, diffusion of PMA into dead bacteria and intercalation into DNA is optimal at 30°C for 15 min in the dark prior to crosslink. After DNA extraction, the target is quantified by real-time PCR, including an IPC for amplification control. SPC in tube 2 serves as calibration standard for 10⁷ bacterial counts, relative to which C_t values can be translated to bacterial counts using the slope of the DNA standard curve. The ΔC_t of tube 2 versus tube 4 tests for sufficient signal reduction from dead cells (including matrix effects and crosslink conditions). If signal reduction from dead cells significantly exceeds the reduction of the signal of the sample in the presence of PMA, the number of “intact and potentially infectious units” (IPIU) can directly be calculated from the C_t value of tube 3, which is translated to bacterial counts as mentioned above.</p

Reduction of dead cell signal of <i>C. jejuni</i> in chicken rinses by PMA.

<p>10⁶–10⁷ dead <i>C. jejuni</i> cells (H₂O₂-treated) were spiked into 1 ml of chicken rinse. Staining was performed either by direct addition of PMA or after centrifugation and resuspension in 1 ml PBS (+ centrifugation). All samples were incubated for 15 min in the dark at the respective temperature before photoactivation. Mean ΔC_t values (with and without PMA) ± standard deviation are shown; n. d., not determined; n, number of tested samples.</p