Research Article: Assessing the occurrence and transfer dynamics of ESBL/pAmpC-producing Escherichia coli across the broiler production pyramid

Date Published: May 17, 2019

Publisher: Public Library of Science

Author(s): Ilias Apostolakos, Lapo Mughini-Gras, Luca Fasolato, Alessandra Piccirillo, Grzegorz Woźniakowski.

http://doi.org/10.1371/journal.pone.0217174

Abstract

Extended-spectrum β-lactamase (ESBL)- and plasmid mediated AmpC-type cephalosporinase (pAmpC)-producing Escherichia coli (ESBL/pAmpC E. coli) in food-producing animals is a major public health concern. This study aimed at quantifying ESBL/pAmpC-E. coli occurrence and transfer in Italy’s broiler production pyramid. Three production chains of an integrated broiler company were investigated. Cloacal swabs were taken from parent stock chickens and offspring broiler flocks in four fattening farms per chain. Carcasses from sampled broiler flocks were collected at slaughterhouse. Samples were processed on selective media, and E. coli colonies were screened for ESBL/pAmpC production. ESBL/pAmpC genes and E. coli phylogroups were determined by PCR and sequencing. Average pairwise overlap of ESBL/pAmpC E. coli gene and phylogroup occurrences between subsequent production stages was estimated using the proportional similarity index, modelling uncertainty in a Monte Carlo simulation setting. In total, 820 samples were processed, from which 513 ESBL/pAmpC E. coli isolates were obtained. We found a high prevalence (92.5%, 95%CI 72.1–98.3%) in day-old parent stock chicks, in which blaCMY-2 predominated; prevalence then dropped to 20% (12.9–29.6%) at laying phase. In fattening broilers, prevalence was 69.2% (53.6–81.3%) at the start of production, 54.2% (38.9–68.6%) at slaughter time, and 61.3% (48.1–72.9%) in carcasses. Significantly decreasing and increasing trends for respectively blaCMY-2 and blaCTX-M-1 gene occurrences were found across subsequent production stages. ESBL/pAmpC E. coli genetic background appeared complex and bla-gene/phylogroup associations indicated clonal and horizontal transmission. Modelling revealed that the average transfer of ESBL/pAmpC E. coli genes between subsequent production stages was 47.7% (42.3–53.4%). We concluded that ESBL/pAmpC E. coli in the broiler production pyramid is prevalent, with substantial transfer between subsequent production levels.

Partial Text

Extended spectrum cephalosporins (ESCs) are considered critically important antimicrobials (CIAs) in both human and veterinary medicine [1,2]. Resistance to these antimicrobials is therefore a major public health concern due to the risks of therapy failure [3]. Resistance in Enterobacteriaceae, such as Escherichia coli, caused by production of extended-spectrum β-lactamases (ESBLs) or AmpC β-lactamases (pAmpCs) and transferability of resistance mechanisms are of particular importance, as the encoding genes (hereafter ESBL/pAmpC genes) are often located in promiscuous plasmids [4]. This property of EBSL/pAmpC genes enables their exchange between bacteria, including pathogens, and favours transmission between animals and humans [5]. Usage of ESCs in poultry has been restricted since 2012 in the European Union (EU) [6] except for occasional use in (grand)parent hatcheries [7,8]. However, numerous studies have shown high prevalence rates of ESBL/AmpC-producing E. coli (ESBL/pAmpC E. coli) in broiler flocks across Europe [9–11], even in countries with low antimicrobial use in livestock [7,12]. The presence of ESBL/pAmpC E. coli has been evidenced at all levels of the broiler production pyramid from breeding farms [13], hatcheries [14], fattening farms [15], slaughterhouses [16] to retail meat [17]. Whether broiler meat contaminated with ESBL/pAmpC E. coli represents an important source of human infections is debatable [18]. For instance, a recent comparative risk assessment estimated the exposure to ESBL/pAmpC E. coli through consumption of chicken meat to be lower than beef and pork [19], whereas a meta-analysis identified poultry products as the most likely source of human ESC-resistant infections [20]. The zoonotic transmission potential has prompted many investigations, not only on the prevalence of ESBL/pAmpC E. coli, but also on their transmission routes along the broiler production pyramid. A recent review [21] summarised the transfer of ESBL/pAmpC-producing bacteria in four major pathways; vertical transmission from parent to offspring, transmission in the hatchery, horizontal transmission in fattening farms, and horizontal transmission between farms or from the environment. Additionally, the authors stressed the need for more quantitative data on these transmission pathways.

This is the first study investigating the occurrence and potential transfer of ESBL/pAmpC E. coli across the whole broiler production pyramid in Italy. Samples predominantly originated from one Italian region, which is however among the most densely populated poultry areas in the country and thus can be considered representative of a large fraction of Italy’s poultry production [30]. Analysis of 820 samples from three broiler production chains resulted in the identification of ESBL/pAmpC E. coli in all farms and batches of carcasses, with an overall prevalence of 60.3%. Day-old PS chicks showed the highest prevalence, comparable with recent findings from the Netherlands [31], but higher than those found in Norway [7]. All ESBL/pAmpC E. coli found in PS chicks had the AmpC phenotype, carried blaCMY-2, and belonged predominantly to phylogroup B2. Therefore, a prevalent E. coli blaCMY-2 lineage seems to have been introduced through the import of day-old PS chicks. However, due to the widespread occurrence of blaCMY-2 in poultry, other sources of entry cannot be excluded [32]. It has been speculated that the underlying reason for high prevalence at this production level is the in ovo use of cephalosporins in supplying (grand)parent hatcheries, which selects for ESC-resistant E. coli in the gut of young hatchlings [8]. This is a problem for Nordic countries supplied by the same breeding stock, as the blaCMY-2 genotype introduced at the top of the production pyramid in these countries spreads clonally to the bottom, is often the only identified ESBL/pAmpC gene [7,33,34]. Similar findings have been described in Denmark [12]. In contrast, we found a significant decrease of blaCMY-2 and a gradual substitution by mainly blaCTX-M-55 and blaCTX-M-1 along the production pyramid, as well as the sharp reduction of prevalence in PS breeders (Fig 1). A similar decrease in the prevalence of blaCMY-2-carrying E. coli from 95% in week 1 to 0% in week 21 has been described by Dame-Korevaar et al. [31] and was attributed to a selective disadvantage of blaCMY-2-carrying plasmids. Although different EBSL/pAmpC genotypes were identified during the laying period, blaCMY-2 was still dominant and the overlap of genotype distributions between the two sampling stages in breeders was substantial despite the relatively long period between them (36.4–50.4%). Persistence of blaCMY-2 has been previously associated with the IncK and IncI1 plasmid types [11].

 

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http://doi.org/10.1371/journal.pone.0217174

 

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