Over the past 20 years, Campylobacter infection have been the most commonly reported zoonosis in EU member states (EFSA and ECDC, 2024) and in the United Kingdom (ACMSF, 2019; UKHSA, 2024). European Food Safety Authority (EFSA) estimated that there were 9 million cases of campylobacteriosis annually in the EU leading to the disease burden of 0.35 million disability-adjusted life years (EFSA, 2011). In the United Kingdom, the estimates are that Campylobacter kills 100 people each year, with case numbers rising since 2005 and annual economic burden being around £900 million (out of a total of around £1.5 billion for all foodborne infections) (FSA, 2013). Most human cases are mild and self-limiting, but in the immunosuppressed, pathogenesis can be further complicated and patients may require treatment i.e., due to Guillan-Barré Syndrome (Igwaran and Okoh, 2019; WHO, 2015) or potentially even die (Holland et al., 2020). The impacts of this infection on quality of life, healthcare and productivity can be therefore substantial.

Approximately 90% of all Campylobacter cases are believed to be caused by C. jejuni, and to lesser extent by C. coli (Liu et al., 2022). Source attribution studies have identified chicken meat as the most frequent cause of Campylobacter infections (Cody et al., 2019). In the UK the estimate is that about 70% of C. jejuni and just under 50% of C. coli human infections have been linked to the chicken source (McCarthy et al., 2021). Whilst most cases of human infection with Campylobacter are treatable, there is a rising concern of antimicrobial resistance, with the WHO ranking fluoroquinolone-resistant Campylobacter as “priority pathogens” (WHO, 2017). Because of this, there has been a change in choice of treatment with macrolides taking preference over the historic use of quinolones (Bolinger and Kathariou, 2017). In Europe, it is mandatory to monitor antimicrobial resistance in food producing animals, food and humans (ECDC, 2016).

It is very well known that the initial carcass contamination occurs during abattoir processing and the prevalence of Campylobacter on carcasses is highest immediately post abattoir processing (Buncic et al., 2017). This is not surprising, since C. jejuni load in caeca can reach 10⁹ CFU/g (Hermans et al., 2011; Wagle et al., 2020) and gut spillage during defeathering and evisceration process can be a very frequent event (Buncic et al., 2017). To control Campylobacter at slaughter, Process Hygiene Criteria (PHC) at abattoir level have been used since 2018. Weekly testing of 5 samples (each sample made of 3 neck skins) is performed over a 10-week period, and no more than 15/50 samples (or 30%) is allowed to exceed 1,000 CFU/g (3 log₁₀) for satisfactory result (from 1st January 2025 no more than 10/50 samples (or 20%) (EC, 2017). The results indicate or verify whether the food business operators’ (FBO) production processes and their Hazard Analysis and Critical Control Points (HACCP) plan are effectively complying with the regulatory framework (Cegar et al., 2022). Furthermore, these results based on the testing for indicator bacteria can be used to compare the performance between abattoirs for the purpose of their risk categorization (Cegar et al., 2022; Salines et al., 2023). Beside Campylobacter, testing for E. coli and Enterobacteriaceae, common commensal indicator microorganisms, has been proposed for the purpose of PHC (Cegar et al., 2022; EFSA, 2012). In their study, Cegar et al. (2022) proposed satisfactory, acceptable or unsatisfactory values for PHC for Enterobacteriaceae, which were 1 log₁₀ higher than the existing values for carcasses of pigs, in order to address inherently “dirtier” chicken slaughter and dressing process (Buncic et al., 2017). These proposed values were m = 4 log₁₀ CFU/g and M = 5 log₁₀ CFU/g, where values over ten sampling sessions (usually a 10-week period) giving a result of ≤ m were deemed satisfactory, values between m and M were considered acceptable, and if values were > M the results for that abattoir was unsatisfactory indicating the needs for improvements in hygiene and process controls.

Abattoir interventions are commonly used at slaughter and some of them have been proven very effective in reducing microbiological contamination (Antic et al., 2021; Zdolec et al., 2022). A substantial number of physical interventions to reduce the prevalence and numbers of Campylobacter on carcasses during slaughter, such as inside-outside carcass wash, ultrasound combined with steam, rapid carcass surface cooling, etc, have been used in poultry industry in recent times, with more or less success (Gichure et al., 2022). These interventions aim to ensure that the FBOs succeed in meeting PHC and produce carcasses with very low Campylobacter load (Buncic et al., 2017).

This study was conducted to evaluate the impact of key abattoir process steps on the levels of Campylobacter spp. and Enterobacteriaceae on chicken carcasses, including decontaminating effect of an innovative non-chemical abattoir intervention combining hot water carcass immersion and ultrasound with final forced dry air chilling. Furthermore, antimicrobial susceptibility of Campylobacter spp. isolates was investigated for antibiotics prioritized as key targets of stewardship programs and monitoring (WHO, 2019a).

This study demonstrated that the abattoir processing substantially decreased Campylobacter spp. and Enterobacteriaceae levels, from the baseline point of defeathering to the final chilled carcass ready for packaging, by 1.14 log₁₀ and 1.43 log₁₀, respectively. The major contributing step in this was intervention based on the combined effect of hot water immersion and ultrasound, which decreased Campylobacter levels by 0.85 log₁₀ and Enterobacteriaceae levels similarly by 0.82 log₁₀ (but with no statistical significance). Forced dry air chilling was effective in reducing Enterobacteriaceae levels by 0.93 log₁₀ (p < 0.05), but ineffective in reducing Campylobacter (0.25 log₁₀, p > 0.05). Evisceration expectedly did not have major effects on contamination, with levels of Enterobacteriaceae only slightly increasing but not significantly by 0.33 logs, indicating that this processing step was properly performed in this abattoir, i.e., machinery calibrated in such a way to avoid gut spillage and contamination. While direct comparisons with other published studies are often difficult due to variable conditions between abattoirs, data available in the literature often show similar trends, with reduction in contamination during scalding, then significant increase during defeathering, usually no change or very small increase in contamination as a consequence of evisceration, and certain reductions achieved during chilling (Buncic et al., 2017). Similar impact of abattoir processing on the levels of Campylobacter spp. and Enterobacteriaceae on final broiler carcasses was also observed in studies by Hauge et al. (2023) (reductions of 1.0 log₁₀ and 1.1 log₁₀, respectively) and Emanowicz et al. (2021) (reduction of 0.8 log₁₀ for Campylobacter). On contrary, reductions of less than 0.5 log₁₀ were reported in studies by Zweifel et al. (2015), Althaus et al. (2017), and Roccato et al. (2018) for both Campylobacter spp. and Enterobacteriaceae. This abattoir used scalding system where the water was changed only once a day, which could imply a possible build-up of contamination during the day and expected increase in microbial load on the carcasses processed last on the day. However, our findings show that some batches processed last on the day (e.g., batches 2 and 3) had lower Campylobacter load than those processed earlier in the day (e.g., batches 4, 5, and 6), which indicated no clear carcass contamination pattern. There may have been other contamination sources for the carcasses on the slaughterline, which could have been investigated by sampling the machinery and environment, but this was outside the scope of our study.

One of the aims in this study was to investigate the effectiveness of the intervention implemented in this abattoir and to our knowledge, this is the first study to present data on hot water immersion and ultrasound intervention for chicken carcasses. Two other similar studies (Moazzami et al., 2021; Musavian et al., 2014) investigated effect of ultrasound combined with hot steam, the SonoSteam system, where steam was used at 90–94°C and ultrasound at 30–40 kHz for 15–20 min, with pooled effect of 1.25 log₁₀ reduction in Campylobacter found in meta-analysis conducted on five trials presented in those two studies (Gichure et al., 2022). In a more recent study, Musavian et al. (2022) found 0.8 log₁₀ reduction for Campylobacter in neck skin samples achieved with the SonoSteam. Reported reduction effects on Enterobacteriaceae with the SonoSteam were similar to Campylobacter in three published studies and were 0.6 log₁₀ (Moazzami et al., 2021) and 1.1 log₁₀ (Musavian et al., 2022). All these reported reductions are in line with our study, and the intervention usually has a little to no effect on the organoleptic features on the final product (Lauteri et al., 2023; Musavian et al., 2014). This strongly suggests that this physical intervention can be recommended for use in chicken abattoirs. Potable water-based interventions that do not use chemicals are considered acceptable by the industry and consumers, as their implementation does not require specific approval by the regulatory authorities (Antic et al., 2021). Indeed, the EU Regulation 853/2004 specifies that the FBOs must not use any substance other than potable water to remove surface contamination from products of animal origin and allows, in principle, the use of other decontamination treatments following appropriate consideration and a risk assessment by EFSA and approval by the regulatory authorities (EC, 2004).

The initial reason for implementing the hot water immersion and ultrasound intervention in this abattoir was to contribute to overall reduction of Campylobacter levels on final chicken carcasses, enabling this FBO to meet the PHC. Testing within the PHC framework is intended as an objective verification measure in the HACCP system allowing abattoirs to take some actions and improve process hygiene. Indirectly, the results of this testing can also be used to communicate the level of Campylobacter risk to consumers purchasing chicken meat processed in abattoirs, in the current absence of food safety criteria for Campylobacter. Even though this intervention achieved significant reduction effects on Campylobacter, the final Campylobacter levels on carcasses post-chilling during the sampling window of 7 weeks exceeded the regulatory limits, indicating that the level of hygiene in this abattoir during this period was unsatisfactory based on the current PHC. Very high levels of Campylobacter were observed on the final carcasses in some weeks, e.g., in weeks 4, 5, and 6, from 3.5 log₁₀ CFU/g to 3.7 log₁₀ CFU/g. This, alongside with the observed higher initial Campylobacter levels post-defeathering (likely reflecting on-farm status), suggests that the reduction capacity of the hot water immersion and ultrasound intervention was possibly exceeded. No single intervention has been shown to be capable of eliminating Campylobacter and a “multiple hurdle” approach would be necessary to produce carcasses with low Campylobacter levels and consequently reduce campylobacteriosis cases in humans (ACMSF, 2019; Goddard et al., 2022). Nevertheless, if Enterobacteriaceae counts were used as indicator microorganism in PHC testing, this abattoir would have achieved a satisfactory result and met PHC based on the limits proposed by Cegar et al. (2022) and Hauge et al. (2023). These indicators are suited for PHC as they indicate fecal contamination and are always present in the process. During the sampling window, contamination with both Campylobacter and Enterobacteriaceae was gradually increasing and peaking in weeks 4–6 after which contamination started decreasing in week 7 when our study finished. This fluctuation in contamination is expected and the FBO must ensure to conduct the trend analysis of the data, and root cause analysis to investigate the problem and rectify it as soon as possible. As Cegar et al. (2022) and Hauge et al. (2023) noted, testing for both Campylobacter and Enterobacteriaceae can be used to risk categorize abattoirs and compare the performances between different abattoirs. In our study, this abattoir would likely be categorized as low-risk based on the Enterobacteriaceae results, and medium-risk based on (mostly borderline) Campylobacter results. However, based on our observations and impression, it is more likely that in the long run, proactive attitude of abattoir’s management in implementing innovative abattoir intervention and overall good slaughter hygiene, would place this abattoir under low-risk category.

Antimicrobial susceptibility of Campylobacter spp. isolates was also investigated in this study for antibiotics prioritized as key targets of stewardship programs and monitoring. Campylobacter isolates were tested for resistance against aminoglycosides (gentamicin and streptomycin), macrolides (erythromycin), quinolones (ciprofloxacin and nalidixic acid) and tetracycline, among which three of these are in the WHO AWaRe Classification Database Watch group (ciprofloxacin, streptomycin, erythromycin) and two in the Access group (gentamycin, tetracycline) (WHO, 2019a). These antimicrobials are either critically important (macrolides, quinolones, aminoglycosides) or highly important for public health (tetracycline) (WHO, 2019b). As expected, high levels of resistance to ciprofloxacin (39%) and tetracycline (45%) were found. This was broadly in line with previous UK surveys of retail chicken meat between 2015 and 2020 where ciprofloxacin resistance in C. jejuni ranged between 41 and 54% and tetracycline resistance between 52 and 68% (Fearnley and Rodgers, 2023; Jorgensen et al., 2021). In the most recent UK study by Fearnley and Rodgers (2023), ciprofloxacin resistance was detected in 65.4% of C. jejuni isolates and tetracycline resistance in 73.8% in chicken meat retail samples, which is higher than in our study. These authors noted that the prevalence of resistance to these antibiotics is not declining in C. jejuni on poultry meat, despite the successful efforts of the British Poultry Council to antimicrobial stewardship scheme to reduce the antimicrobial use in poultry meat chain (Fearnley and Rodgers, 2023; UKVARSS, 2022). Resistance to erythromycin, streptomycin and gentamicin was very low (2, 1, and 1%, respectively), which was also in line with previous UK studies (Fearnley and Rodgers, 2023; Jorgensen et al., 2021). Multidrug resistance, which is characterized as the acquired ability to be resistant to at least one antimicrobial agent in three or more different classes (Magiorakos et al., 2012), was found in only 2.7% of Campylobacter isolates. However, there were four multidrug resistant Campylobacter isolates that were co-resistant to both ciprofloxacin and erythromycin (plus tetracycline), in 1.6% of samples, that had very high counts of 700 CFU/g, 5,000 CFU/g, 5,500 CFU/g, and 7,500 CFU/g. This is of a potential public health concern because erythromycin is the main treatment option for complex cases of campylobacteriosis and both ciprofloxacin and erythromycin are classified as a highest priority critically important antimicrobial (HP-CIA) by the WHO (WHO, 2019b).

All batches except for batch 5 exhibited clear antimicrobial resistance to at least one antibiotic with 48.7% Campylobacter isolates found to be resistant overall. Batches 1, 2, and 6 had very high resistance to tetracycline of 84, 97, and 98%, respectively. Of these, batch 1 and batch 2 had both a history of disease and antibiotic treatment with Doxycycline hyclate, a tetracycline antibiotic. This study was also a part of a wider research of antimicrobial resistance in broiler meat production chain by Abdulla (2024), where the same neck skin samples were analyzed for antimicrobial resistant and extended-spectrum beta-lactamase (ESBL)-producing E. coli. The author found that 57.4% of the E. coli isolates and 25% of ESBL-producing E. coli isolates were also resistant to tetracyclines, and 22.2% of E. coli isolates and 100% of ESBL-producing E. coli isolates were resistant to ciprofloxacin (Abdulla, 2024). Given their broad spectrum nature and affordability, tetracyclines are the second most often used class of antibiotics in the British poultry sector (UKVARSS, 2022). Tetracyclines are considered highly important for public health (WHO, 2019b) and should be used with prudence (European Medicines Agency, 2019).

As a result of the importance of Campylobacter in the disease burden, many countries have adopted national surveillance and control systems that have not always been proved fully successful. For example, in the United Kingdom, the Food Standards Agency (FSA) began a Campylobacter reduction program in 2010 in collaboration with the industry. The overall goal was to reduce the proportion of chicken meat highly contaminated with Campylobacter at retail (> 1,000 CFU/g), which would result in significant reduction (15–30%) in the number of human cases as shown by modeling (FSA, 2010, 2015). A recent study compared European Campylobacter surveillance programs and found that 12 European countries, with the exception of France, demonstrated a decline in human reported Campylobacter cases since the PHC for Campylobacter was implemented in 2018 (Olsen et al., 2024). However, this is not the case in the UK where human cases have remained stable at around 100 per 100,000 population between 2014 and 2019 (UKHSA, 2024), even though the proportion of chicken meat samples at retail with greater than 1,000 CFU/g of Campylobacter dropped from 19 to 5% (and have remained stable since then) (Kintz et al., 2024). To investigate the continued high incidence in human cases, the UK FSA commissioned an expert elicitation exercise to generate testable hypotheses that might explain why the reduction in highly contaminated chicken carcasses available at retail did not correspond to decreased levels of human disease (Kintz et al., 2024). In total, 25 hypotheses were generated, but the prevailing opinion was that the reasons were likely complex and multifactorial. Notable reasons were that the Campylobacter infectious dose could be lower than assumed 1,000 CFU, which can also be linked to increased chances of infection in the UK aging and vulnerable population. There is also a possibility of an increase in the presence of Campylobacter virulent strains in the food as a consequence of selective pressure from interventions used in the food chain. Insufficient and/or inadequate controls on farm and during slaughter and processing may select more resistant strains that may be more virulent for humans, offsetting the reductions achieved in Campylobacter prevalence in chicken meat at retail (Kintz et al., 2024). In our study, we did not investigate the virulence characteristics of Campylobacter isolates, but we found that 2.7% of Campylobacter isolates were associated with multidrug resistance, among which four isolates co-resistant to ciprofloxacin, erythromycin and tetracycline, in samples with very high Campylobacter counts of up to 7,500 CFU/g. The presence of multidrug resistant strains alongside high number of Campylobacter counts on final carcasses may be of a potential public health concern.