Neck Skin Samples Research Articles

Harmonizing Campylobacter risk assessments across European countries – can the pooled process hygiene criteria data be used in the Danish risk assessment model?

This study investigated the possibility of harmonizing quantitative microbiological risk assessment (QMRA) for Campylobacter spp. across European (EU) countries. French Campylobacter data (2020–2021) from neck skin (NS) pools, sampled at slaughterhouses under the European surveillance component for Food Business Operators (FBOs), were adapted to inform a QMRA model that, among others, has been used within the Danish Action Plan against Campylobacter, on the basis of single leg skins (LS) data. Datasets included culture results (in colony forming unit per gram, CFU/g) from 1,284 broiler flocks slaughtered at 13 slaughterhouses representing broiler production in western France. Five pools (of 2–4 NS samples each) per flock were tested. One pool per tested flock was randomly chosen for the analysis. After conducting descriptive statistics (on flock prevalence and meat contaminations across months and years), three contamination transformation factors (CTFs) were estimated to translate NS pools contaminations into single LS contamination, based on data from French and Danish studies. A reference simulation scenario (ScenRef) was set with CTF = 3.2 (i.e. NS pool concentration divided by 3.2); while other 13 scenarios represented an alternative scenario analysis to investigate the impact of: the CTF value (ScenMin with CTF = 2 and ScenMax with CTF = 10), censored test results (ScenUncens) and random choice of pool per flock (ScenSampling-1 to 10), on the risk estimates. The average monthly/annual risk of human disease per poultry meal and the monthly/annual relative risk (RR) of 2021 compared to 2020, were estimated. In ScenRef, the annual RR was 1.22, suggesting an increase of risk of ≈ 22 % in 2021 compared to 2020. The impact of CTFs, censored data and randomized pool sampling per flock, on the annual and (most) monthly RRs, appeared limited. This study gives an overview of the strengths and limitations to be considered for adapting the French FBO data into the Danish model and to harmonize risk assessments across EU countries, accordingly. To reduce uncertainty in risk estimates, it could be considered increasing representativeness of NS tested flock populations and/or using LS rather than NS samples; because LS samples are more representative of actually retailed meat contaminations. If NS pools are maintained, the relationships between concentrations on NS pools and those on consumed meat requires further investigation.

Persistence of Salmonella and Campylobacter on Whole Chicken Carcasses under the Different Chlorine Concentrations Used in the Chill Tank of Processing Plants in Sri Lanka.

The persistence of non-typhoidal Salmonella and Campylobacter in chicken meat is a considerable public health risk and a future challenge. This study aimed to determine the prevalence of Salmonella and Campylobacter in poultry processing lines where different chlorine concentrations were used in the chill tank. The samples were collected from four types of processing plants in Sri Lanka, considering the chlorine concentration used in the chill tank, which ranged from 2 ppm to 50 ppm. Salmonella and Campylobacter were isolated from whole carcass washings, neck skin, and cecal samples. Subsequently, an antimicrobial susceptibility test was performed for the isolates. The results revealed the overall prevalence of Salmonella and Campylobacter was 78.25% and 63.5%, respectively. Positive percentages of Salmonella and Campylobacter were high in the carcasses compared to the neck skin and ceca. The Campylobacter counts on the whole carcasses were significantly low (p < 0.001), at higher chlorine concentrations ranging from 20 to 30 ppm and 40 to 50 ppm. The pathogen prevalence in the whole carcasses was 84.7% Campylobacter coli, 39.1% Campylobacter jejuni, 71.1% Salmonella Typhimurium, and 28.8% Salmonella Infantis. The highest resistance was observed for tetracycline (63.8%) in Salmonella, while it was for gentamicin (87.8%) in Campylobacter. The prevalence percentage of multidrug-resistant Campylobacter was 51.2%, while it was 2.12% for Salmonella. The persistence of multidrug-resistant Salmonella and Campylobacter on the post-chill carcasses was highlighted in the present study as a significant public health threat that has to be addressed urgently.

Investigation of the Resistance of Some Disinfectant Active Substances in ESBL-Producing Enterobacteriaceae

In this study, a total of 200 samples, including 100 neck skin and 100 cecum samples, were collected and analyzed from various poultry slaughterhouses on different sampling days. ESBL-producing Enterobacteriaceae were isolated and ESBL production was confirmed phenotypically by combined disk diffusion and E-test gradient strips. While ESBL production was confirmed in 10 (10%) of 100 neck skin samples, no significant ESBL production could be confirmed in 100 cecal samples. The broth microdilution method of Clinical and Laboratory Standards Institute (CLSI) was used to determine the resistance profiles against benzalkonium chloride (BC), cetylpyridinium chloride (CPC), N-alkyl dimethyl benzyl ammonium chloride (ADBAC) and potassium peroxymonosulfate (PPMS) disinfectants in 10 neck skin isolates with confirmed ESBL production. In the study, it has been determined that MIC50 and MIC90 values were respectively ADBAC (8 and 16 mg/L), BC (16 and 32 mg/L), CPC (16, and 32 mg/L), PPMS (≥ 1024 mg/L). The impacts of Enterobacteriaceae strains on food safety and public health are significant; Disinfectant resistance can lead to increased transmission of antibiotic-resistant bacteria, leading to serious infections in humans that are difficult to treat. For that reason, it is of great importance to develop effective control methods, including appropriate disinfectant use, hand hygiene and appropriate isolation measures, to prevent the spread of disinfectant resistant Enterobacteriaceae strains in food production systems.

Implementation of a national monitoring programme of Campylobacter in Irish broilers to measure progress of on-farm and primary processing control measures.

Campylobacter is the most common food-borne pathogen in the European Union. In 2018, the crude incidence rate in Ireland was 63.6 per 100,000 population. Chicken is considered an important source of infection for humans. In 2015, the Campylobacter Stakeholders' Group (CSG) was established to reduce Campylobacter contamination levels in Irish broiler flocks. This work aimed to describe the Campylobacter monitoring programme that was established by the CSG, to analyse the results of this testing between 2019 and 2022, and to assess progress. This paper describes the monitoring programme that was established by the CSG, which harmonized Campylobacter enumeration testing across all Irish broiler processors and allowed comparability of results for trend analysis. An analysis of the 2019-2022 data is presented here and compared to previous studies of Campylobacter levels in Irish broilers. An analysis of the 2019-2022 data showed a significant reduction in levels in both caeca and neck skin when the results from 2022 were compared to those from 2019 to 2020. Campylobacter spp. were detected in 37% of caecal samples from first depopulation (pre-thin) broilers and 30% of neck skin samples in 2022, with just 4% of carcases (in neck skin samples) with ≥1000 colony-forming units per gram detected in 2022. Campylobacter levels detected in Irish broilers, in the present monitoring programme were less than those reported in previous studies in both caecal and carcase samples, although not directly comparable for statistical significance because of differences in study methods. The cooperation between stakeholders and regulators of the Irish broiler chicken industry over the past decade has facilitated a coordinated approach to monitoring of Campylobacter levels in broilers, and implementation of control measures. This has enabled a steady reduction in the levels of Campylobacter in Irish chicken.

Whole genome sequence-based characterization of Campylobacter isolated from broiler carcasses over a three-year period in a big poultry slaughterhouse reveals high genetic diversity and a recurring genomic lineage of Campylobacter jejuni

Campylobacter is among the most frequent agents of bacterial gastroenteritis in Europe and is primarily linked to the consumption of contaminated food. The aim of this study was to assess genomic diversity and to identify antimicrobial resistance and virulence genes of 155 Campylobacter isolated from broiler carcasses (neck skin samples) in a large-scale Swiss poultry abattoir over a three-year period. Samples originated from broilers from three different types of farming systems (particularly animal-friendly stabling (PAFS), free-range farms, and organic farms).Campylobacter jejuni (n = 127) and Campylobacter coli (n = 28) were analysed using a whole genome sequencing (WGS) approach (MiniSeq; Illumina). Sequence types (STs) were determined in silico from the WGS data and isolates were assigned into complex types (CTs) using the cgMLST SeqSphere+ scheme. Antimicrobial resistance genes were identified using the Resistance Gene Identifier (RGI), and virulence genes were identified using the virulence factor database (VFDB).A high degree of genetic diversity was observed. Many sequence types (C. jejuni ST19, ST21, ST48, ST50, ST122, ST262 and C. coli ST827) occurred more than once and were distributed throughout the study period, irrespective of the year of isolation and of the broiler farming type. Antimicrobial resistance determinants included blaOXA and tet(O) genes, as well as the T86I substitution within GyrA. Virulence genes known to play a role in human Campylobacter infection were identified such as the wlaN, cstIII, neuA1, neuB1, and neuC1. Subtyping of the Campylobacter isolates identified the occurrence of a highly clonal population of C. jejuni ST21 that was isolated throughout the three-year study period from carcasses from farms with geographically different locations and different farming systems.The high rate of genetic diversity observed among broiler carcass isolates is consistent with previous studies. The identification of a persisting highly clonal C. jejuni ST21 subtype suggests that the slaughterhouse may represent an environment in which C. jejuni ST21 may survive, however, the ecological reservoir potentially maintaining this clone remains unknown.

High Salmonella load with serovar virchow dominance pose major public safety risk in postchill broiler carcasses

The objective of this study was to determine Salmonella contamination levels, presence and serovar distribution in broiler carcasses before and after chilling, as well as to evaluate the effectiveness of chilling process. A total of 96 pooled neck skin samples (PNSS) of 48 prechill (PreC) and 48 postchill (PosC) carcasses, representing 480 broilers collected in 6 mo' period were analyzed using ISO 6579-2:2012 Miniaturized Most Probable Number (ISO-mMPN) technique. Species confirmation and serovar identification was performed by Salmonella-specific real-time PCR (Salm-PCR) and conventional serotyping, respectively. Mean Salmonella count was 1.84 log10 MPN/g in PreC, and 1.48 log10 MPN/g in PosC samples, indicating a statistically significant reduction of 0.36 log10 MPN/g (p < 0.05) in the counts by plant's air chill system. Salmonella positivity reduced from 97.9% (47/48) in PreC to 85.42% (41/48) in PosC samples, confirmed by Salm-PCR with identified serovars as S. Virchow (89.77 %) followed by S. Schwarzengrund (9.09%) and S. Bredeney (1.14%). Persistence of high load and prevalence of Salmonella with serovar Virchow dominance (other than the ones mandated in current guidelines) in the final product contributes significant and up to date data to relevant literature, and provides unbiased epidemiological reference to legal authorities for future relevant revisions.

Status quo: Levels of Campylobacter spp. and hygiene indicators in German slaughterhouses for broiler and turkey

Poultry is a common reservoir for Campylobacter and a main source for human campylobacteriosis. With broiler being the predominant poultry for food production, most food safety related research is conducted for this species, for turkey, few studies are available. Although animals are typically colonized at the farm level, the slaughtering process is considered an important factor in re- and cross-contamination. We examined the development of Campylobacter, E. coli and total colony counts (TCC) after several processing steps in three broiler and one turkey slaughterhouses. Whole carcass rinsing and neck skin sampling was applied for broilers resulting in 486 samples in total, while 126 neck skin samples were collected for turkeys. A decrease in the loads of the different bacterial groups along the broiler slaughtering process was observed. Campylobacter mean counts dropped from 4.5 ± 1.7 log10 CFU/ml after killing to 1.6 ± 0.4 log10 CFU/ml after chilling. However, an increase in Campylobacter counts was evident after evisceration before the values again decreased by the final processing step. Although the Campylobacter prevalence in the turkey samples showed a similar development, the bacterial loads were much lower with 1.7 ± 0.3 log10 CFU/g after killing and 1.7 ± 0.4 log10 CFU/g after chilling compared to those of broilers. The loads of E. coli and total colony count of turkey were higher after killing, were reduced by scalding and remained stable until after chilling.This study highlights trends during the slaughtering process in reducing the levels of Campylobacter, E. coli, and total colony counts for broiler and turkey carcasses, from the initial step to after chilling. These results contribute to our understanding of microbial dynamics during meat processing.

Process Hygiene Criterion for Campylobacter and Number of Campylobacter Enteritis Cases in Northwest Germany.

Campylobacteriosis is the most commonly reported bacterial foodborne disease in the European Union. Its transmission is often associated with the consumption of poultry meat. In 2018, Regulation (EC) No. 2017/1495 introduced a process hygiene criterion and with this, the testing requirements for Campylobacter. The results of microbiological testing for Campylobacter of chicken carcass neck skin samples from several slaughter lines in Northwest Germany collected by the food business operators and contamination levels (cfu/g Campylobacter) of these samples were analysed from 2018 to 2021. Classification into three different categories was made based on contamination levels. The proportion of highly contaminated (category three) neck samples (>1000 cfu/g) decreased from 2018 to 2021. Our analysis showed a relationship between the number of neck samples with high Campylobacter contamination levels (>1000 cfu/g) and human cases in Northwest Germany. Spearman's rank test (p < 0.01) showed a higher correlation in 2018 (0.66) and 2019 (0.58) compared to 2020 and 2021. Campylobacter enteritis cases in Northwest Germany stayed at a low level in 2020 and 2021. It remains unclear whether the decrease in reported Campylobacter enteritis cases is related to a decrease in Campylobacter levels on chicken carcasses or due to other reasons like underreporting during the COVID-19 pandemic, and therefore must be investigated in further analyses.

Use of harmonised epidemiological indicators (HEIs) for broilers in Europe

In 2012, the European Food Safety Authority (EFSA) proposed harmonised epidemiological indicators (HEIs) for poultry at different production stages: Salmonella, Campylobacter, extended-spectrum β-lactamase/AmpC β-lactamase (ESBL/AmpC) producing Enterobacteriaceae and generic Escherichia (E.) coli. The HEIs are based on existing monitoring systems or the sampling strategies provided by EFSA. To realise the full benefit of HEIs, risk managers should use them for farm and abattoir categorisation and for adapting the existing meat inspection systems. As HEIs are not legal requirements within the European Union (EU), it is unclear which HEIs are used in which country, to date. Therefore, an online survey was conducted in Europe to gather knowledge about the implementation, application and consequences following on from the HEIs in existing official and private monitoring and surveillance systems (MOSS).A total of 34 answer sets from participants working in the framework of official surveillance or as food business operators in broiler abattoirs were collected from eleven EU member states (EU-MS) and four non-EU countries.While all participants stated that testing for Salmonella is performed, HEI 4-Salmonella, which corresponds to the process hygiene criterion (PHC) for Salmonella was applied by 62% of the participants. In total, 94% of the participants reported that they test for Campylobacter. Among them, 71% stated that testing is performed for HEI 5-Campylobacter, which corresponds to PHC for Campylobacter. Although testing neck skin samples for Salmonella and Campylobacter after chilling are official and mandatory MOSS in the EU, not all participants from EU-MS (Salmonella: 6/11 EU-MS; Campylobacter: 8/11 EU-MS) confirmed to comply with. Altogether, 56% of the participants (from 6 EU-MS and 2 non-EU countries) stated that they test for E. coli. Ten of them reported that the testing is performed at the abattoir after chilling according to the suggested HEI for generic E. coli as a hygiene indicator. Consequences that result from the existing MOSS for the three examined pathogens (Salmonella, Campylobacter and E. coli) were mainly rising awareness, farm risk categorisation and feedback to the farmer.According to the answers from the participants, the HEIs suggested by EFSA for broilers are currently implemented in most EU-MS. One reason could be that some of the according MOSS are required by EU law. As intended by EFSA, the participants stated that they use HEIs for farm risk categorisation as one of the three top consequences following from MOSS for the three mentioned pathogens. For improving the knowledge and application of HEIs in the context of risk-based meat safety assurance systems, specific training could be helpful.

The Slaughterhouse as Hotspot of CC1 and CC6 Listeria monocytogenes Strains with Hypervirulent Profiles in an Integrated Poultry Chain of Italy.

In Europe, very few studies are available regarding the diversity of Listeria monocytogenes (L. monocytogenes) clonal complexes (CCs) and sequence types (ST) in poultry and on the related typing of isolates using whole genome sequencing (WGS). In this study, we used a WGS approach to type 122 L. monocytogenes strains isolated from chicken neck skin samples collected in two different slaughterhouses of an integrated Italian poultry company. The studied strains were classified into five CCs: CC1-ST1 (21.3%), CC6-ST6 (22.9%), CC9-ST9 (44.2%), CC121-ST121 (10.6%) and CC193-ST193 (0.8%). CC1 and CC6 strains presented a virulence gene profile composed of 60 virulence genes and including the Listeria Pathogenicity Island 3, aut_IVb, gltA and gltB. According to cgMLST and SNPs analysis, long-term persistent clusters belonging to CC1 and CC6 were found in one of the two slaughterhouses. The reasons mediating the persistence of these CCs (up to 20 months) remain to be elucidated, and may involve the presence and the expression of stress response and environmental adaptation genes including heavy metals resistance genes (cadAC, arsBC, CsoR-copA-copZ), multidrug efflux pumps (mrpABCEF, EmrB, mepA, bmrA, bmr3, norm), cold-shock tolerance (cspD) and biofilm-formation determinants (lmo0673, lmo2504, luxS, recO). These findings indicated a serious risk of poultry finished products contamination with hypervirulent L. monocytogenes clones and raised concern for the consumer health. In addition to the AMR genes norB, mprF, lin and fosX, ubiquitous in L. monocytogenes strains, we also identified parC for quinolones, msrA for macrolides and tetA for tetracyclines. Although the phenotypical expression of these AMR genes was not tested, none of them is known to confer resistance to the primary antibiotics used to treat listeriosis The obtained results increase the data on the L. monocytogenes clones circulating in Italy and in particular in the poultry chain.

Prevalence and Antimicrobial-Resistant Patterns of Non-typhoidal Salmonella in Good Agricultural Practice Certified Broiler Farms and Poultry Slaughterhouses in an Intensive Farming Area in Upper Northern Part of Thailand

Non-typhoidal Salmonella (NTS) is still one of the most infectious foodborne pathogens causing problematic health issues worldwide in both human and veterinary medicine. Poultry meat was one of the important sources of NTS spreading and tended to be highly resistant to antibiotics. The aim of this study was to determine the prevalence, serotypes, and antimicrobial-resistant patterns of Salmonella circulating in broiler farms and poultry slaughterhouses in an intensive farming area in the upper northern part of Thailand from August to October 2019. Fifty samples of boot swabs were collected from 50 broiler farms, 50 cecal samples, and 250 neck skin samples from slaughterhouses. Salmonella was identified by culture method and serum-agglutination and antimicrobial susceptibility testing was tested using the automated VITEK-2 compact system. This study's total prevalence of Salmonella was 53.71% (n=188/350). We found that 53% (159/300) of cecal and neck skins samples were collected from slaughterhouses and 58% (29/50) of boot swabs collected from broiler farms were positive for NTS. Twenty-four serotypes of NTS were identified, the most encountered was S. Kentucky. The antimicrobial-resistant patterns showed that all the strains were non-susceptible to amikacin, cefalexin, cephalothin, and gentamicin and were susceptible to imipenem, neomycin, and nitrofurantoin. The NTS prevalence in samples from broiler farms was slightly higher than in poultry slaughterhouses indicating that there was contamination in the farming and slaughtering process. Therefore, both hygienic measurements in poultry production and antimicrobial usage in the poultry industry should be considered.

Forensic application of epidermal expression of HSP27 and HSP70 for the determination of wound vitality in human compressed neck skin

Estimating the age and vitality of human skin wounds is essential in forensic practice, and the use of immunohistochemical parameters in this regard remains a challenge. Heat shock proteins (HSPs) are evolutionarily conserved universal proteins that protect biological systems from various types of stress. However, its importance in forensic pathology for determining wound activation in neck compression skin remains unclear. The expression of HSP27 and HSP70 in neck skin samples was immunohistochemically examined to understand its forensic applicability in determining wound vitality. Skin samples were obtained from 45 cases of neck compression (hanging, 32 cases; strangulation, 10 cases; manual strangulation, 2 cases; other, 1 case) during forensic autopsies; intact skin from the same individual was used as a control. HSP27 expression was detected in 17.4% of keratinocytes in the intact skin samples. In the compressed region, the frequency of HSP27 expression in keratinocytes was 75.8%, which was significantly higher than that in intact skin. Similarly, HSP70 expression was 24.8% in intact skin samples and 81.9% in compressed skin samples, significantly higher in compressed skin than in intact skin samples. This increase in case compression cases may be due to the cell defence role of HSPs. From a forensic pathology perspective, the immunohistochemical examination of HSP27 and HSP70 expression in neck skin could be considered a valuable marker for diagnosing traces of antemortem compression.

The microbiological and sensory status of dual-purpose chickens (Lohmann Dual), male Lohmann Brown Plus chickens, and conventional laying hens slaughtered in a laying hen abattoir compared to conventional broilers slaughtered in a broiler abattoir.

Alternatives to conventional chicken meat and egg production are increasingly under discussion, especially because of the common practice of killing male day-old chicks from laying lines which has been banned from the beginning of 2022 in Germany and is planned to be banned during 2022 in other countries. Production of dual-purpose chicken lines is one possible solution, as such lines combine moderate laying and growth performance. The microbiological status of products from such breeds must be comparable to existing products on the market for food safety purposes. Additionally, the production of such products will take longer because of the feeding regimes required, and again, comparability should be safeguarded for the best consumer protection. The dual-purpose chicken line, Lohmann Dual (males), was compared to males from the laying line Lohmann Brown Plus, conventional laying hens (all slaughtered and processed in the same conventional laying hen abattoir), and conventional broilers (slaughtered in a conventional broiler abattoir). Neck skin samples were taken before chilling at the end of each slaughter line to determine the microbial status of the carcasses. Additionally, fresh and cooked meat sensory analysis was performed on meat from broilers and male and female Lohmann Dual and Lohmann Brown Plus chickens (for three carcasses of each group) at the German Agricultural Society Test Center in Kassel. The focus was on the performance of male Lohmann Dual compared to the other lines. There was no difference in the Enterobacteriaceae count of the dual-purpose chicken line compared to conventional broilers, whereas laying hens had a significantly higher microbial load before chilling, as based on neck skin examinations (p&lt;0.001). According to sensory test results, the meat from dual-purpose chickens was the best (as no defects were found) among the five chicken meat types examined. In conclusion, based on their microbial status and sensory analysis of fresh and cooked meat, Lohmann Dual males slaughtered in a laying hen abattoir can be considered as an alternative to conventionally kept broilers slaughtered in a broiler abattoir.

Genomic diversity, virulence and source of Campylobacter jejuni contamination in Irish poultry slaughterhouses by whole genome sequencing.

The aim was to exploit whole genome sequencing (WGS) to assess genomic diversity, identify virulence genes and deduce the proportion of Campylobacter colonized broilers that directly contaminate their carcasses. Campylobacter jejuni isolates (107) from caeca and carcass neck skin samples (50 pairs from the same batch plus 7 individual caeca) sampled at three poultry slaughterhouses over a one-year period were selected for sequencing (MiSeq; Illumina). FastQ files were submitted to BioNumerics for analysis using the wgMLST scheme for allele calling. Campylobacter cgMLST and hierarchical clustering was performed by applying the single linkage algorithm. Sequence types (STs) were determined in silico from the WGS data and isolates were assigned into clonal complexes (CCs) using the Campylobacter PubMLST.org database. Virulence genes were determined by downloading core sequences from the virulence factor database (VFDB) and the National Center for Biotechnology Information (NCBI). A high degree of diversity was observed with 23 different STs identified. ST257 and CC-21 were the most common STs and CCs, respectively. cgMLST analysis suggested that 56% of carcass contamination was a direct result of contamination from caeca from the same batch. Virulence genes known to play a role in human C. jejuni infection were identified such as the wlaN gene and the genes associated with lipooligosaccharide synthesis, which were identified in 30% of isolates. Caecal colonization was the more plausible occurring source of C. jejuni contamination of broiler carcasses, compared with cross-contamination from another batch or the environment. The high rate of genetic diversity observed amongst caecal isolates is consistent with a wide variety of Campylobacter strains circulating in poultry flocks in Ireland. The results will further inform broiler processors and regulators about the influence and importance of on-farm colonization versus slaughterhouse cross-contamination and the relationship between C. jejuni in caeca and carcasses during processing.

Quantitative assessment of Campylobacter spp. levels with real-time PCR methods at different stages of the broiler food chain

The importance of thermotolerant Campylobacter spp. as a food-borne pathogen continues to increase and there is a great need for rapid quantitative results in routine diagnostics. However, currently, only the culture-based ISO method is authorized for use in the context of official food control. The present study therefore aimed to assess the suitability of a qPCR method for a rapid quantitative determination of Campylobacter spp. at different stages in the poultry production chain and its equivalence with the culture-based method. Samples from two processors were collected and evaluated both separately and together. Censored regression (Tobit) models have been used to establish a relationship between Campylobacter qPCR counts on the carcasses and explanatory variables of processor and meat counts. Further, correlations of qPCR Campylobacter spp. counts at the different stages of production were calculated. In addition, the comparative data between microbiological enumeration and qPCR results were statistically analyzed. In the correlation calculation of the qPCR results, a highly significant relationship between the Campylobacter spp. counts of the neck skin samples to breast fillet and leg samples could be calculated, indicating a good prediction of Campylobacter spp. loads in these samples.The intercalating dye ethidium monoazide (EMA) was used to see whether the correlations between microbiological counts and qPCR results were improved by pretreating fecal and cecal samples before qPCR analysis. It was shown that the observed values of scatter plots between the qPCR-based and the culture-based methods were strongly correlated. However, on average, the qPCR results were two log10 CFU/mL levels higher than the microbiological counts. However, the classical culture-based method for food hygiene risk assessment cannot be replaced one-to-one by the qPCR or EMA-qPCR. The qPCR method can rather be used for the rapid identification of particularly highly contaminated flocks.

Efficacy of peroxyacetic acid against Salmonella biofilms and as a decontaminant agent in poultry meat

We aimed to investigate the effect of the post-chilling Peroxyacetic Acid (PAA) application on chicken carcasses’ shelf life, PAA treatment on chicken neck skin samples contaminated with S. Enteritidis and S. Typhimurium serotypes, and its activity against single and mixed Salmonella biofilms. Initially, effects of the post-chilling PAA application on chicken carcasses’ shelf life was analyzed. Secondly, the effectiveness of PAA application in chicken neck skin samples which are experimentally contaminated was investigated. Then, biofilm forming capacity of Salmonella enterica serovars were determined, and PAA activity against single and mixed Salmonella biofilms was evaluated. According to the results, in all experimental groups at least 1 log cfu/g reduction was observed on the day of PAA application (0 hour), and we monitored significant decreases of Salmonella counts in all groups after both 100 and 200 ppm PAA treatments within the 6th hour. The combinations of PAA concentrations and duration of application used in the study were insufficient in terms of S. Typhimurium and S. Enteritidis counts in chicken neck skin samples; however, these treatments can be effective on extending the chicken meat’s shelf life. On the other hand, 200 ppm of PAA was effective in reducing S. Typhimurium and S. Enteritidis biofilms. As a mean of 4.8 log/ml reduction were detected against S. Enteritidis and S. Typhimurium biofilms. Moreover, we can conclude from statistical analysis that when biofilm is composed with more than one Salmonella, it is harder to reduce their counts with PAA treatments. However, there is a recommendation of EFSA (European Food Safety Authority) that PAA may be a decontamination agent in poultry production in the future, considering that PAA will not have toxic effects on human health. PAA studies on this subject will be of great importance in the future if it is passed to legislation.

Comparative Study of Fresh and Frozen Broiler Neck Skin Sampled for Process Hygiene Purposes

The objective of the study was to determine the effect of freezing broiler neck skin samples before their microbial analysis, compared to freshly examined samples regarding total viable count (TVC) and Enterobacteriaceae count (EC). For this, 300 neck skin samples were taken at a German commercial broiler abattoir and each neck skin sample was cut into two parts. One randomly selected part underwent microbial examination after storage at 4 °C overnight; the other part was frozen at −30 °C for eight weeks before analysis in the same laboratory. Log cfu/g values of TVC and EC were separately compared between the fresh and frozen neck skin samples. A difference up to 0.5 log values was set as acceptable, i.e., fresh and frozen samples with counts that differed by this amount were considered as not different. The differences between the grouped samples of fresh and frozen broiler neck skin regarding both TVC and EC levels were less than 0.5 log values. Thus, it can be assumed that broiler neck skin samples, both fresh and frozen for eight weeks, are suitable for microbiological examination, as the TVC and EC results showed equivalence. Therefore, freezing broiler neck skin samples can be an option to maintain viable bacteria levels in broiler neck skin samples taken for microbiological examination in process control, when freezing and later examination is necessary due to insufficient laboratory capacity for the examination of fresh neck skin samples.

Antibiotic, heavy metal, and disinfectant resistance in chicken, cattle, and sheep origin E. coli and whole‐genome sequencing analysis of a multidrug‐resistant E. coli O100:H25 strain

AbstractIn this study, antibiotic, heavy metal, and disinfectant resistance profiles of E. coli and E. coli O157 isolates were determined, and whole‐genome sequencing of a multidrug‐resistant E. coli O100:H25 strain was reported. A total of 70 E. coli isolated from chicken neck skin and sheep cecum samples, and 32 E. coli O157 strains isolated from cattle carcass, sheep carcass, and slaughterhouse wastewater samples were explored. Minimum inhibitory concentrations (MICs) of one antibiotics, eight heavy metals, and three disinfectants were determined by the broth microdilution method. Twenty of those isolates exhibiting high MIC values against compounds tested were further analyzed by PCR for the presence of relevant resistance genes (n = 42). The majority of the isolates tested were resistant to erythromycin and/or fosfomycin (99% and 78%, respectively) and 89% of the isolates were multidrug‐resistant. Among the heavy metals, and disinfectants that were quaternary ammonium compounds (QAC) tested, the highest prevalence of resistance was observed against nickel (71%) and followed by zinc (62%), and N‐alkyl‐dimethyl‐benzyl‐ammonium chloride (26%). While blaAmpC, ermC, murA, and aadA were the most abundant antibiotic resistance genes, rcnA and zntA, and mdfA, sugE (c), and ydg(F) were also commonly observed as heavy metal and disinfectant genes, respectively. Additionally, whole‐genome sequencing was performed for a single multidrug‐resistant strain (E. coli P91). This strain was identified as serotype O100:H25, and harbored three Inc class plasmids and ant(2″)‐Ia, aph(3′)‐Ia, aph(3″)‐Ib, aph(6)‐Id, blaTEM‐1A, dfrA5, mdf(A), sul1, sul2, and tet(A) genes along with the various heavy metal and disinfectant related genes. The findings of the study show that both phenotypic and genotypic antibiotic, heavy metal, and disinfectant resistance are highly prevalent in E. coli isolates that originated from food‐producing animals. The use of antimicrobials in food‐producing animals needs to be carefully evaluated since the coexistence of antibiotic, heavy metal, and disinfectant resistance genes may result in a coselection that yields the emergence and spread of highly persistent and resistant strains in agricultural settings.

Antimicrobial resistance and genomic diversity of Campylobacter jejuni isolates from broiler caeca and neck skin samples collected at key stages during processing.

Phenotypic antimicrobial resistance testing of 160 Campylobacter jejuni isolates from broiler caeca and neck skin samples collected at key stages during processing from 32 batches was conducted to: (i) determine the prevalence of antimicrobial resistance in C. jejuni isolates (ii) investigate if C. jejuni strains with particular resistance profiles were more frequently recovered at the end of processing, (iii) to establish if multiple strains with different resistance profiles were present within individual batches of broilers and (iv) to determine the virulence profiles of isolates. C. jejuni isolates were tested for resistance to erythromycin (ERY), ciprofloxacin (CIP), tetracycline (TET), gentamicin (GEN), nalidixic acid (NAL) and streptomycin (STR) using a broth dilution phenotypic method. Resistance to TET was most prevalent (46%), followed by resistance to CIP and NAL (29%). All isolates were susceptible to ERY and GEN, while resistance to STR was low (2%).

Prevalence and levels of Campylobacter in broiler chicken batches and carcasses in Ireland in 2017–2018

In 2008, an EU wide baseline survey of broilers revealed a high Campylobacter prevalence. To assist with industry-wide controls, updated data were required. The primary objective of this study was to establish up-to-date data on Campylobacter carriage and carcass contamination in Irish broilers. Monthly samples were collected from the three largest broiler processing plants in Ireland over a twelve-month period. Samples were taken from both first and final thin birds (partial and full depopulation) from 358 batches of broilers. From each batch, a composite sample of 10 caecal contents (n = 358) and 5 neck skins (n = 1790) were collected and numbers of Campylobacter in each sample were determined. Of the 1790 neck skin samples tested, 53% were Campylobacter positive. Campylobacter was detected in the caecal contents of 66% of all batches tested. Depopulation and/or age had a significant effect on Campylobacter prevalence with 67% of final thin broilers yielding Campylobacter-positive neck skin samples in contrast to 38% of first thin broilers that yielded positive neck skin samples (P ≤ 0.002). A significant seasonal variation was observed in the rate of Campylobacter-positive caecal samples with higher prevalence seen in July (85%) than the colder months of November (61%), December (50%), January (61%) March (57%) and April (59%). Neck skin samples were 7 times more likely to be Campylobacter positive if the caecal contents from the same batch were positive (odds ratio = 7.1; P ≤ 0.0001). The decrease in Campylobacter prevalence observed in neck skin and caecal contents demonstrates the improvements and progress made in reducing prevalences of this important enteropathogen in the Irish poultry industry since the 2008 EU baseline survey. It also provides further supporting data on the impact of thinning, the processing environment and season on Campylobacter prevalence.