Dynamics of CMY-2 producing E. coli in a broiler parent ﬂ ock

Extended-spectrum β -lactamase and plasmid mediated AmpC β -lactamase (ESBL/pAmpC) producing bacteria are resistant to Extended Spectrum Cephalosporins (ESC), and are present in all levels of the broiler production chain. We determined the prevalence, concentration, and persistence of ESBL/pAmpC- Escherichia coli in a broiler parent ﬂ ock during the rearing and laying period. One-day old chickens were housed in four separate pens. Until week 33 no antibiotics or coccidiostatics were used. During rearing 57 chickens in each pen ( n = 228), and in the laying period two groups of 33 chickens were individually sampled ( n = 66). Environmental samples were taken from week 16 onwards. ESBL/pAmpC- E. coli presence was determined by selective culturing. In the samples of week 16 – 19 the concentration of ESBL/pAmpC- E. coli was determined. All ESC-resistant isolates found were positive for pAmpC gene bla CMY-2 located on IncA/C plasmids, in several E. coli MLST types. CMY-2- E. coli prevalence decreased from 91% (95%CI 86 – 94%) at day 7 (week 1) to 0% (95%CI 0 – 5%) in week 21. However, CMY-2- E. coli remained present in the environmental samples during the whole study. CMY-2- E. coli concentration varied between detection limit (<10^3) and 2·10^4 cfu/g faeces. The sharp reduction of CMY-2- E. coli in this broiler parent ﬂ ock in absence of antibiotics suggests a selective disadvantage of bla CMY-2 on IncA/C plasmids on animal level. The underlying mechanism should be studied further as this may provide new insights on how to reduce ESBL/pAmpC prevalence and transmission in the broiler production chain.


Introduction
Extended-spectrum β-lactamase and plasmid mediated AmpC βlactamase (ESBL/pAmpC) producing bacteria are resistant to Extended Spectrum Cephalosporins (ESC). In the Netherlands, 56.5% of the broilers at slaughter were carriers of ESBL/pAmpC-Escherichia coli in 2015 (MARAN, 2016). Although prevalence in poultry varies between farms (Blaak et al., 2015), ESBL/pAmpC producing bacteria are present in all levels of the broiler production chain (Dierikx et al., 2013). The broiler production chain has a pyramidal structure, thus the presence of ESBL/pAmpC in the upper levels of the chain might influence the ESBL/ pAmpC status of lower levels in the chain, e.g. through vertical transmission (Nilsson et al., 2014;Zurfluh et al., 2014). To our knowledge reports on the dynamics of ESBL/pAmpC-E. coli in parent stock are lacking. The aim of this study is to determine prevalence, faecal concentration, and persistence of ESBL/pAmpC-E. coli in a broiler parent flock during the rearing and laying period.

Chickens
One-day old broiler parent stock chickens (n = 3184) were housed in a rearing house of an experimental poultry farm in the Netherlands. The chickens were divided over four completely separated pens. Each pen housed 693 females and 103 males, separated by a fence. At week 20 all chickens were moved to the laying house. Two groups of 30 females and three males were selected from the four rearing pens, and randomly allocated to two separate pens. During the laying phase two females died and one lame male was replaced in pen 1 and one female died in pen 2.
Chickens received feed without antibiotics or coccidiostats. Feed and water were available ad libitum during the first seven days, thereafter feed was supplied based on body weight. Drinking water pipes were cleaned before entry of the chickens and thereafter weekly by acidifying the water using peracetic acid. Chickens received a microflora product (Aviguard ® ) at day of arrival. A standard vaccination and lighting scheme was applied.

Identification and sampling
2.2.1. Rearing period At day 7 (week 1), in each pen 57 females (minimal sample size to detect 5% prevalence) were selected randomly, and sampled by individual cloacal swabs. At week 12 per pen 57 females were randomly selected, tagged and individual cloacal swabs were taken. In week 16-19 the tagged females were sampled weekly and environmental samples were taken using bootsocks. In week 19 environmental samples were taken in the male pens.

Laying period
At week 20, the females and males were moved to the laying house. All females found ESBL/pAmpC-E. coli positive at least once during week 16-19 and a random selection of females being ESBL/pAmpC-E. coli positive in week 12 were selected and housed in two groups of 30 females. To each group three randomly selected and tagged males were added. All chickens were sampled individually in week 21, 24, 34 and 35. Environmental samples from the litter were taken at week 21, 24, 34 and 35 (morning) and 43, 45, 46, 47 and 49 (noon). In week 34 and 35 also environmental samples from the laying nests were taken, by hand wiping using bootsocks. In week 49 chickens were euthanized and caecal content was collected.

Antibiotic treatment
In week 33 the chickens in pen 1 were administered amoxicillin via the drinking water for five days (20 g/1000 kg live weight/day).

Follow up offspring
During week 34, 160 eggs were collected from both pens and disinfected with formaldehyde. Forty eggs were crushed, eggshells and egg content was mixed and analysed for ESBL/pAmpC presence, 120 eggs were incubated. After hatching, individual cloacal swabs were taken from the broilers at day of hatch, daily until day 7 and at days 14 and 21. Environmental samples were taken at the same days, starting the day after hatch. At day 21 broilers were euthanized and caecal content was collected.

Ethics
The animal procedures at Utrecht University were approved by the Animal Ethical Committee of Utrecht University (Utrecht, the Netherlands), in full compliance with all relevant legislation.
2.6. Analysis 2.6.1. ESBL/pAmpC-E. coli detection Cloacal and caecal samples, eggs and bootsocks were selectively cultured (3 mL LB broth versus 400 mL LB, supplemented with 1 mg/L cefotaxime). After overnight incubation at 37°C, 10 μL broth was inoculated on MacConkey plates supplemented with 1 mg/L cefotaxime and incubated overnight at 37°C. Cloacal samples were analysed individually. Eggs and caecal samples were pooled per five, bootsocks were pooled per pen, except for bootsocks taken in week 16 and 43-49.

ESBL/pAmpC-E. coli and E. coli concentration
Swabs used in week 16-19 in pen 1 and 2 were weighed before and after sampling to determine the amount of faeces collected. Swabs were suspended in 1 mL saline solution and tenfold dilution series were made to quantify the colony-forming units (cfu) of ESBL/pAmpC-E. coli and total E. coli per mL, using MacConkey plates with and without 1 mg/L cefotaxime. Based on the amount of faeces on the swabs cfu/gram faeces was calculated.

Typing
From week 12 onwards, in at least one isolate of every sampling moment, ESBL/pAmpC genes were typed by PCR and sequencing (Dierikx et al., 2010). Plasmids were characterized by transformation (Dierikx et al., 2010) and PCR-based Replicon Typing (PBRT) (Diatheva, Italy). Selection of transformants was performed on LB agar containing 1 mg/L cefotaxime. E. coli genotyping was performed by MLST (Wirth et al., 2006). MLST patterns were analysed using Bionumerics version 6.1.
The high prevalence in week 1 might be the result of vertical transmission from the grandparent flock or other sources of contamination at the hatchery or during transport. The grandparent flock had been treated with antibiotics in the weeks prior to production of the parent stock. Unfortunately, no data about the ESBL/pAmpC prevalence in this flock is available. Despite the high prevalence at day 7, CMY-2-E. coli was not able to persist in the chickens. Other studies in poultry have also shown a decreasing prevalence of antibiotic resistant bacteria, with and without the use of antibiotics (Diarra et al., 2007;Baron et al., 2014;Huijbers et al., 2015). However, most of these studies report limited reduction. Factors as ageing (Lu et al., 2003) (Stanley et al., 2012) and stress (Burkholder et al., 2008) might influence the microbiota composition and thus the potential of CMY-2-E. coli to persist in the gut. Until week 33 no antibiotics were used, resulting in no selective advantage to CMY-2-E. coli. After applying amoxicillin in week 33 in one of the pens, 2/31 chickens became positive in week 34, in the non-treated pen prevalence was 0%. However, one week later, 1/32 chickens was positive in the non-treated pen, whereas no positive samples were found in the treated pen (Fig. 1). The inability to persist on animal level might be due to an unsuccessful combination of CMY-2-E. coli on plasmid IncA/C. In European broiler meat, CMY-2-E. coli is often found in combination with plasmids IncI1 or IncK (Borjesson et al., 2013;Egervarn et al., 2014). The low occurrence and the observed decrease in this study may suggest that plasmid IncA/C is less able to conjugate and spread in bacterial populations as was previously described for Salmonella (Poole and Crippen, 2009).
Contrary to the decreasing prevalence and CMY-2-E. coli concentration in the faeces, almost all environmental samples (89/116) were positive (Fig. 1). Before placement of the chickens the laying house tested negative for ESBL/pAmpC-E. coli. Based on the positive environmental samples, negative cloacal swabs during the laying period and negative caecal samples at the end of the experiment, the chickens most likely introduced CMY-2-E. coli into the laying pens and after that ceased shedding CMY-2-E. coli. Environmental contamination might have persisted after the birds ceased excretion. Others report survival of E. coli and ESBL-E. coli in faeces and soil for months (Merchant et al., 2012). Although CMY-2-E. coli was still present in the environment during egg collection, none of the samples taken from the eggs and offspring were found positive for ESBL/pAmpC-E. coli.

Conclusion
This study showed that in a parent flock at an experimental farm, in absence of antibiotics, prevalence of pAmpC gene bla CMY-2 on IncA/C plasmid decreased and is not detected in the offspring. This may not be true for other farms, with different ESBL/pAmpC-plasmids in E. coli, and under field conditions. The mechanism behind this should be studied further as this might lead to possible interventions to reduce ESBL/pAmpC prevalence and transmission in the broiler production chain.

Funding
This work was supported by Public Private Partnership project: 1H4F-Reduction of ESBLs: evaluation of ESBL interventions (TKI-AF-14210).

Conflicts of interest
None.