Quantification of ESBL-Escherichia coli on broiler carcasses after slaughtering in Germany

doi:10.1016/j.fm.2015.10.020

Food Microbiology

Volume 54, April 2016, Pages 1-5

https://doi.org/10.1016/j.fm.2015.10.020 Get rights and content

Highlights

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Thirty broiler flocks from German slaughterhouses were enumerated for ESBL-Escherichia coli.
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Caecal contents and neck skin samples as surrogate for the meat were analysed.
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ESBL-E. coli had high detection rates but low counts in the neck skin samples.
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There was no correlation of total E. coli to ESBL-E. coli in caeca or in neck skin.

Abstract

Extended spectrum beta lactamase forming Escherichia coli is considered a threat in severe clinical cases. Food producing animals and poultry in particular are considered a possible reservoir for distribution of resistant bacteria to humans via meat consumption. Data on the concentration of ESBL-E. coli as a prerequisite for estimating consumer exposure is still limited. To evaluate the amount of ESBL-E. coli present in meat of broilers after slaughtering we sampled pooled caecal contents and pooled neck skin samples as meat surrogate of 30 broiler flocks for commensal and ESBL-E. coli at three German slaughterhouses. Although ESBL-E. coli were present in 90% of the flocks caeca, they only made up a fraction of the total E. coli (mean 7.9 log₁₀ cfu/g), the ratio varying with a median value of 0.07%. Samples of neck skin were positive for ESBL-E. coli in 93.3% of the flocks. However analyses in neck skin pooled samples, revealed countable ESBL-E. coli in only 32.7% (n = 150) of the samples with concentrations between 1.0 and 3.1 log₁₀ cfu/g. In conclusion, concentrations of ESBL-E. coli seem to be low in this study, thus indicating a limited impact of broiler meat for ESBL-E. coli transmission to humans via consumption.

Introduction

Escherichia coli producing extended spectrum β-lactamases (ESBL) inactivate β-lactam antibiotics and broad spectrum cephalosporins of class 3 (Livermore, 2008). Additional resistance genes confer resistance to a wide range of different antimicrobials. As a result, ESBL-producers are considered a serious threat for therapy failure in human medicine and are increasingly reported during the last decade (Livermore, 2009). ESBL-producing E. coli were shown to be prevalent in several food animal species at farm level and in products thereof (Dierikx et al., 2010, Geser et al., 2011). Possible associations between human and broiler ESBL-E. coli were reported and broilers were proposed as a potential reservoir for human acquisition of foodborne resistant bacteria, because analysed strains of human and poultry origin shared similar ESBL-genes or plasmid types (Ewers et al., 2012, Kluytmans et al., 2013). Little is known about the number of ESBL-E. coli on carcasses after slaughtering at processing level which is the first step in meat production. Quantification is of interest when it comes to risk assessments or feasibility of potential intervention measures (Ellerbroek, 2012). It is currently being discussed to develop microbiological criteria or performance targets for antimicrobial resistant bacteria at the broiler meat production level to aid food safety (EFSA, 2012a). Enumeration studies are needed in advance to understand the current situation and to evaluate the status-quo. This will help to quantify the impact of the broiler meat production chain on consumer exposure. The aim of this study was to assess the number of ESBL-producing E. coli on market ready broilers in Germany collected at the end of slaughtering in 3 different processing facilities. This is also an example how sampling for ESBL-E. coli could be implemented in the existing microbiological hygiene control system at slaughterhouses.

Section snippets

Material and methods

In this study a total of 30 flocks were sampled after slaughtering at three different slaughterhouses (A–C), with 10 flocks sampled per slaughterhouse. The flocks, a unit of broilers of the same age and from the same house of a farm, were representative of conventional flocks, the size of the flocks ranging from 30,000 to 40,000. Per flock 10 caeca were collected, with 300 samples in total for the 30 flocks. Caeca content is considered to be representative for the amount of bacteria introduced

Caeca samples

A total of 90% of the 30 flocks tested positive for ESBL-E. coli in pooled caeca samples after enrichment. The three individual slaughterhouses A, B and C had prevalence rates for each of the 10 tested flocks of 80%, 90% and 100%, respectively (Table 1). In slaughterhouses A and B the ESBL-E. coli test positive flocks in addition had values above the limit of enumeration in all samples, with means of 4.5 cfu/g and 4.9 cfu/g, respectively. In slaughterhouse C, 7 out of the 10 positive tested

Discussion

Extended spectrum β-lactamase gene carrying E. coli were found in broiler flocks of all three slaughterhouses with high percentages at the end of the slaughtering process. The results were within the range of reports from other studies which found ESBL-E. coli in ca. 40% up to >90% of broiler meat at the retail level (Egea et al., 2012, Kola et al., 2012, Overdevest et al., 2011). Nevertheless, the high prevalence of ESBL-E. coli does not coincide with high bacterial numbers. Only 32.7% of the

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Cited by (12)

Enumeration, antimicrobial resistance and genomic characterization of extended-spectrum β-lactamases producing Escherichia coli from supermarket chicken meat in the United Arab Emirates
2023, International Journal of Food Microbiology
The occurrence and counts of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli in retail chicken sold in the United Arab Emirates (UAE) were investigated in this study. Results indicated that 79.68 % of chicken carcasses (251/315) sampled from UAE supermarkets harbored ESBL-producing E. coli. About half (51.75 % [163/315]) of the tested samples had an ESBL-producing E. coli count range between ≥3 log₁₀ and < 5 log₁₀ CFU/g. The antimicrobial resistance profiles of a subset of 100 isolates showed high rates of non-susceptibility to clinically significant antibiotics, particularly ciprofloxacin (80 %) and cefepime (46 %). Moreover, 7 % of the isolates exhibited resistance to colistin, with PCR-based screening revealing the presence of the mcr-1 gene in all colistin-resistant isolates. Multiplex PCR screening identified bla_CTX-M and bla_TEM genes as the most frequently presented genes among the phenotypically confirmed ESBL-producing E. coli. Further whole-genome sequencing and bioinformatic analysis of 27 ESBL-producing E. coli isolates showed that the gene family bla_CTX group 1 was the most prevalent, notably CTX-M-55 (55.55 % [15/27]), followed by CTX-M-15 (22.22 % [6/27]). The most common sequence types (STs) were ST359 and ST1011, with three evident clusters identified based on phylogenomic analysis, aligned with isolates from specific production companies. Analysis of plasmid incompatibility types revealed IncFIB, IncFII, Incl2, and IncX1 as the most commonly featured plasmids. The findings of this study indicate a noticeable prevalence and high counts of ESBL-producing E. coli in chicken sampled from supermarkets in the UAE. The high rates of antimicrobial resistance to clinically important antibiotics highlight the potential public health risk associated with consuming chicken contaminated with ESBL-producing E. coli. Overall, this study emphasizes the importance of continued antimicrobial resistance monitoring in the UAE food chain and calls for further exposure risk assessment of the consumption of ESBL-producing E. coli via chicken meat.
Whole genome sequence analysis of antimicrobial resistance genes, multilocus sequence types and plasmid sequences in ESBL/AmpC Escherichia coli isolated from broiler caecum and meat
2020, International Journal of Food Microbiology
Citation Excerpt :
At the slaughterhouse level, significant differences between slaughterhouses have been observed in terms of the effectiveness of hygienic procedures in ESBL/AmpC E. coli reduction of fecal contamination. Hence, improvements could be attainable (Pacholewicz et al., 2015; Reich et al., 2016). We found that from two farms, ESBL/AmpC E. coli was found from meat packages even when the broiler (caecal) samples from those farms were negative.
Plasmid-encoded extended-spectrum β-lactamase and AmpC gene-carrying Escherichia coli (ESBL/AmpC E. coli) is an increasing cause of human infections worldwide. Increasing carbapenem and colistin resistance further complicate treatment of these infections. The aim of this study was to assess the occurrence of ESBL/AmpC E. coli in different broiler flocks and farms, as well as in broiler meat, in a country with no antimicrobial usage in broiler production. An additional goal was to assess the genetic characteristics of ESBL/AmpC E. coli isolates by using whole genome sequencing (WGS). Altogether 520 caecal swabs and 85 vacuum-packed broiler meat samples were investigated at the slaughterhouse level. WGS of the bacterial isolates revealed acquired antimicrobial resistance (AMR) genes, multilocus sequence types (MLST) and plasmid sequences. ESBL/AmpC E. coli was identified in 92 (18%) of the caecum and 27 (32%) of the meat samples. ESBL/AmpC E. coli-carrying birds derived from six (33%) out of 18 farms. Of the two bla_ESBL/AmpC genes detected by PCR, bla_CMY-2 (96%) was predominant over bla_CTX-M-1 (4%). Furthermore, WGS revealed an additional AMR gene sul2. Carbapenemase, colistin, and other AMR genes were not detected from the isolates of either the caecal or meat samples. Altogether seven MLSTs (ST101, ST117, ST212, ST351, ST373, ST1594 and an unknown ST) and a variety of different plasmid sequences (IncB/O/K/Z, IncI1, IncFII, IncII, IncFIB, IncFIC, IncX1 and an additional set of Col-plasmids) were detected. This is the first study on genomic epidemiology of ESBL/AmpC E. coli on broiler farms and flocks with no antimicrobial usage, by using WGS analysis. Results show that ESBL/AmpC E. coli occurrence is common both in the caecum and in the packaged meat. However, compared to other European countries, the occurrence is low and the presence of AMR genes other than bla_CMY-2 and bla_CTX-M-1 is rare. More studies are needed to understand the ESBL/AmpC E. coli occurrence in broiler production to prevent the meat from contamination during slaughter and processing, thereby also preventing zoonotic transmission of ESBL/AmpC E. coli. Additionally, more studies are needed to understand the ecology and fitness cost of Enterobacteriaceae plasmids in animal production in order to prevent their acquisition of plasmid-encoded antimicrobial resistance genes such as carbapenem and colistin resistance genes, as this would pose a great hazard to food safety.
Impact of selective and non-selective media on prevalence and genetic makeup of ESBL/pAmpC-producing Escherichia coli in the broiler production pyramid
2020, Veterinary Microbiology
Citation Excerpt :
All analyses accounted for clustering of data at the farm-chain level (Apostolakos et al., 2019). To study the correlation between the loads of total E. coli on EMB and putatively ESBL/AmpC growing on CTX-EMB, we followed the methodology of Reich et al. (Reich et al., 2016) and analysed the data with two methods: first, only samples with countable loads on CTX-EMB were included. In a second attempt, E. coli loads of samples negative for the quantitative but positive for the qualitative method on CTX-EMB, were arbitrarily set to 0.9 Log CFU/mL (i.e. just below LOQ) whereas for samples negative for both methods, loads were set to 0.0 Log CFU/mL.
Presence of extended-spectrum β-lactamase (ESBL)- and AmpC β-lactamase (pAmpC)-producing Escherichia coli (ESBL/pAmpC-EC) in humans and animals is alarming due to the associated risks of antibiotic therapy failure. ESBL/pAmpC-EC transmission between the human and animal compartments remains controversial. Using cefotaxime-supplemented (selective) media, we recently showed high sample prevalence of ESBL/pAmpC-EC in an integrated broiler chain [i.e. Parent Stock (PS), offspring broilers and their carcasses]. Here, we used a different approach. In parallel with the selective isolation, samples were processed on non-selective media. E. coli isolates were tested for ESBL/pAmpC-production and those found positive were genotyped. For carcasses, total E. coli were enumerated. This approach enabled us to estimate prevalence at the isolate level, which mirrors ESBL/pAmpC-EC colonisation levels. We showed that although present in many animals, ESBL/pAmpC-EC were overall subdominant to intestinal E. coli, indicating that high sample prevalence is not associated with high levels of resistance in individual hosts. This is a relevant aspect for risk assessments, especially regarding the immediate exposure of farm personnel. An exception was a particularly dominant B2/bla_CMY-2 lineage in the gut of imported PS chicks. This predominance obscured presence of latent genotypes, however bias towards particular ESBL/pAmpC-EC genotypes from the selective method or underestimation by the non-selective approach did not occur. At the slaughterhouse, we showed a link between total E. coli and ESBL/pAmpC-EC on carcasses. Mitigation strategies for reducing consumers’ exposure should aim at suppressing ESBL/pAmpC-EC in the broiler gut as well as controlling critical points in the processing line.
Contamination of chicken meat with extended-spectrum beta-lactamase producing- Klebsiella pneumoniae and Escherichia coli during scalding and defeathering of broiler carcasses
2019, Food Microbiology
Extended-spectrum beta-lactamase- (ESBL-) producing Klebsiella (K.) pneumoniae and Escherichia (E.) coli are of critical importance in human and veterinary medicine. Animal food products, especially broiler chickens, are discussed as a possible source for the exposure of humans with antibiotic resistant bacteria. Although the occurrence and vertical transmission of ESBL-/AmpC-producing Enterobacteriaceae in the broiler production has been reported before, detailed investigations concerning the dissemination along the slaughter processing line are missing.
In this study, we investigated cross-contamination with ESBL-producing Enterobacteriaceae during the processing of two different broiler flocks in one slaughterhouse. The ESBL-status during the fattening period of the flocks was determined and environmental samples from the slaughterhouse were taken before processing of the respective flocks. These isolates were compared to those found in samples from the carcasses after processing using whole genome sequencing. Phylogenetic analyses of seven ESBL-producing K. pneumoniae and 14 E. coli revealed close relationships between isolates from scalding water and the defeathering machine, respectively, which were collected before the processing of the broiler flocks, to those isolates found in samples from skin and filet of the respective flock carcasses. In conclusion, using high resolution molecular data we found evidence for the cross-contamination of carcasses with ESBL-producing Enterobacteriaceae during scalding and defeathering in the slaughterhouse.
Prevalence and quantitative analysis of ESBL and AmpC beta-lactamase producing Enterobacteriaceae in broiler chicken during slaughter in Germany
2018, International Journal of Food Microbiology
Citation Excerpt :
The median of cefotaxime resistant Enterobacteriaceae was 2.5 × 103 cfu/g in caecum, 1.5 × 103 cfu/g in skin and 1.5 × 102 cfu/g in filet samples. A high prevalence of ESBL-/AmpC-producing Enterobacteriaceae did not necessarily result in high concentrations of ESBL-/AmpC-producing Enterobacteriaceae, this was also shown in other studies (Cohen Stuart et al., 2012; Reich et al., 2013; Reich et al., 2016). However, Smet et al. (2011) showed by means of an in situ continuous flow culture system that an avian E. coli strain could proliferate without selective pressure of an antibiotic and that a gene transfer between avian and human E. coli strains could already take place at a concentration of 103 cfu/ml of the donor strain.
Food producing animals are considered a reservoir for Extended-spectrum beta-lactamase (ESBL) and AmpC beta-lactamase (AmpC) producing Enterobacteriaceae. Therefore, meat is discussed to be a potential source for the transmission of these resistant bacteria to humans. There is only limited information about the quantitative load of ESBL-/AmpC-producing Enterobacteriaceae in different sample matrices during slaughter and their distribution in the slaughterhouse environment. Therefore, the aim of this study was to determine the prevalence as well as quantitative load of ESBL-/AmpC-producing Enterobacteriaceae in caecum, skin and filet samples of different broiler chicken flocks during slaughter in Germany. In addition, environmental samples were taken during slaughter of the respective flocks. To gain insights into possible transmission routes of ESBL-/AmpC-producing Enterobacteriaceae, the corresponding phylogroup and beta-lactamase genes were determined for selected isolates.
ESBL-/AmpC-producing Enterobacteriaceae were detected during slaughter of all seven investigated flocks. On average, 47% (83/175) of caecum, 55% (96/175) of skin, 28% (49/175) of filet and 28% (25/89) of environmental samples harboured ESBL-/AmpC-producing Enterobacteriaceae. Prevalence varied widely between the flocks as well as between the different sample matrices. In about half of the caecum (23/40) and skin (19/40) samples as well as 85% (17/20) of the filet samples, the number of putative ESBL-/AmpC-producing Enterobacteriaceae (cefotaxime resistant Enterobacteriaceae) was below quantification limit. The median of cefotaxime resistant Enterobacteriaceae was 2.5 × 10³ cfu/g in caecum, 1.5 × 10³ cfu/g in skin and 1.5 × 10² cfu/g in filet samples. The median of cefotaxime resistant Enterobacteriaceae was, depending on the sample matrix, 1–4 log units below the median of total Enterobacteriaceae. Using real-time PCR, in 82% (629/767) of the cefotaxime resistant Enterobacteriaceae at least one of the investigated beta-lactamase genes bla_CTX-M, bla_SHV, bla_TEM, bla_AmpC-CIT was detected. The respective resistance genes of 322 isolates were further sequenced. The predominant bla-gene was bla_CMY-2 (48%), followed by bla_SHV-12 (23%).
A contamination from the broiler chicken to the slaughterhouse environment and vice versa seems probable as isolates of the same species and phylogroup, encoding the same resistance genes were detected in all matrices during slaughter of the respective flock as well as in the slaughterhouse environment.
Level of Detection (LOD<inf>50</inf>) of Campylobacter Is Strongly Dependent on Strain, Enrichment Broth, and Food Matrix
2022, Frontiers in Microbiology

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Short communicationQuantification of ESBL-Escherichia coli on broiler carcasses after slaughtering in Germany

Highlights

Abstract

Introduction

Section snippets

Material and methods

Caeca samples

Discussion

J. Food Prot.

Int. J. Food Microbiol.

Vet. Microbiol.

Int. J. Food Microbiol.

Clin. Microbiol. Infect.

J. Food Prot.

J. Food Prot.

J. Food Prot.

Food Microbiol.

Clin. Microbiol. Infect.

Int. J. Food Microbiol.

Int. J. Food Microbiol.

Commission Regulation (EC) No 2073/2005 of 15 November 2005 on Microbiological Criteria for Foodstuffs

Short communication
Quantification of ESBL-Escherichia coli on broiler carcasses after slaughtering in Germany