Salmonella spp. and Extended-Spectrum Cephalosporin-Resistant Escherichia coli Frequently Contaminate Broiler Chicken Transport Cages of an Organic Production Company

Abstract

Antimicrobial resistant bacteria in retail meat pose a health hazard to the public, as does contamination of these products with Salmonella. Our aim was to determine the prevalence of Salmonella as well as Escherichia coli expressing AmpC and extended-spectrum beta-lactamase (ESBL) resistance phenotypes contaminating broiler transport cages and fresh, retail ground chicken meat. Sterile gauze sponges were used to collect duplicate cage floor samples from transport trailers that deliver market-ready birds to a single organic poultry-processing facility. With the exception of the first visit (n = 25), 50 duplicate cage floor samples were collected using moistened sterile gauze sponges on each of nine weekly visits during May, June, and July 2013. Additionally, fresh, retail ground chicken meat was sampled at each weekly visit from an on-site retail store located at the same processing facility. A total of 425 cage swabs and 72 ground chicken aliquots from 24 retail packages were collected and screened for the presence of Salmonella as well as E. coli expressing resistance to extended-spectrum cephalosporins using selective culture. We recovered Salmonella from 26.1% of cage swab samples and 2.8% of retail meat samples. E. coli expressing AmpC and ESBL resistance phenotypes were recovered from 84.9% and 22.6% of cage swabs and 77.8% and 11.1% of fresh, retail ground meat samples, respectively. Our results suggest that transport cages could potentially act as a source of broiler exposure to both Salmonella and enteric bacteria resistant to important antimicrobial drugs as they are transported for entry into the food supply as fresh, retail meat products.

Introduction

S almonella infection causes ∼1.2 million illnesses and an estimated 450 deaths annually in the United States (Scallan et al., 2011), and most cases of salmonellosis are the result of foodborne transmission, accounting for 86% of global cases (Majowicz et al., 2010). The Centers for Disease Control and Prevention estimates that Salmonella is the causative agent for 11% of domestically acquired foodborne illnesses and 28% of deaths from foodborne illnesses (Scallan et al., 2011). Chicken meat is an important food vehicle for transmission of Salmonella and has been associated with numerous outbreaks (Jackson et al., 2013; Gieraltowski et al., 2016; Medalla et al., 2016).

Salmonella can be readily recovered from fecal samples from broilers in both organic and conventional operations (Renwick et al., 1992; Bailey and Cosby, 2005; Liljebjelke et al., 2005; Rodriguez et al., 2006; Alali et al., 2010) and from litter drag swabs in broiler barns (Mallinson et al., 1989; Caldwell et al., 1998). In chickens, Salmonella colonization generally does not result in clinical disease (Ishihara et al., 2009). Chicks may be first exposed to Salmonella in the hatchery, and previous studies have reported that up to 9% of day-old chicks are colonized with Salmonella (Bailey et al., 1994, 2001). Shedding of Salmonella from colonized broilers can increase measurably under stress conditions such as transportation (Marin and Lainez, 2009).

The 2014–2015 Retail Meat Interim Report from the U.S. Food and Drug Administration's National Antimicrobial Resistance Monitoring System reported that 9.1% and 6.2% of retail chickens were contaminated with Salmonella spp. in 2014 and 2015, respectively. This contamination rate was the highest among meat products tested, which included turkey, beef, and pork products in addition to chicken (FDA, 2017). Additionally, organic or antibiotic-free production practices do not appear to impact the probability of recovery of Salmonella from retail chicken products when compared with products from conventionally raised broilers (Mollenkopf et al., 2014).

In addition to pathogens, fresh, retail meat products can serve as a vehicle for the zoonotic foodborne transmission of commensal bacteria harboring clinically important antibiotic resistance genes. These bacteria may colonize the enteric flora of consumers following ingestion, serving as potential reservoirs of resistance genes. Broilers are frequently colonized with Enterobacteriaceae harboring extended-spectrum cephalosporin resistance genes on mobile plasmids (Dierikx et al., 2013; Nilsson et al., 2014). Fresh retail chicken meat is also commonly contaminated with Enterobacteriaceae, such as Escherichia coli harboring these same resistance genes (Mollenkopf et al., 2014).

The stress associated with feed withdrawal or transport of broilers to harvest may contribute to an increase in both the shedding of Salmonella by birds already colonized and the colonization of naïve birds (Rigby and Pettit, 1980; Corrier et al., 1999; Marin and Lainez, 2009; Buhr et al., 2017). This suggests that broilers could be exposed to Salmonella and resistant enteric bacteria in previously contaminated transport cages just before slaughter, which could then increase food safety risk. However, little data has been gathered about the level of Salmonella or antibiotic-resistant bacterial contamination in broiler transport cages. Therefore, our objective was to assess the extent to which broiler transport cages may be acting as a source for Salmonella spp. and extended-spectrum cephalosporin-resistant E. coli that could ultimately contaminate the food supply.

Materials and Methods

We utilized a cross-sectional, observational sampling approach to address our objectives. We sampled the floors of empty broiler cages on transport trailers that were used to deliver market-ready birds to a single large, organic poultry-processing facility. The time that had elapsed between the last unloading of the cages and our sampling was not obtained, but all trailers sampled were in regular daily use. With the exception of the first visit (n = 25), 50 cage floor samples were collected on each of 9 weekly visits during May, June, and July 2013. Sampling was divided equally between the trailers that were available at the time of collection. Samples consisted of two separate sterile gauze sponges moistened with sterile buffered peptone water used concurrently on equal portions of the same transport cage floor. The first swab was used to screen for Enterobacteriaceae harboring beta-lactamase genes conferring resistance to clinically important extended-spectrum cephalosporins. The second gauze sponge was used to screen for Salmonella, a common foodborne pathogen. Both gauze sponges were placed into sterile Whirl-Paks for transport to the laboratory for processing.

For the culture of extended-spectrum cephalosporin-resistant Enterobacteriaceae, the gauze was incubated overnight at 37°C in 36 mL of MacConkey broth modified with 2 μg/mL cefotaxime. A sterile swab was used to inoculate three plates of MacConkey agar, one modified with 4 μg/mL cefoxitin, one modified with 4 μg/mL cefepime, and one with 1 μg/mL meropenem to select for the AmpC, extended-spectrum beta-lactamase (ESBL), and carbapenem-resistant phenotypes, respectively. All plates were streaked for isolation, incubated overnight, and checked for growth. An isolated lactose-fermenting colony was picked from each positive growth, tested for indole production, and later preserved at −80°C in 30% glycerol. DNA was extracted through a boiled lysate protocol (Johnson and Nolan, 2009) for confirmation of the presence of bla _CMY (AmpC) and bla _CTX-M (ESBL) genes responsible for conferring resistance to extended-spectrum cephalosporins. Identification of bla _CMY (AmpC) and bla _CTX-M (ESBL) genes was performed using standard polymerase chain reaction (PCR) techniques. Primers used for bla _CMY amplification were forward primer AmpC1 (5′-ATGATGAAAAAATCGTTATGC-3′) (Winokur et al., 2001) and reverse primer CMY_1038R (5′-TACGTAGCTGCCAAATCCACCAGT-3′) (Kanwar et al., 2013). Forward primer PANCTX-M.F (5′-TTTGCGATGTGCAGTACCAGTAA-3′) and reverse primer PANCTX-M.R (5′-CGATATCGTTGGTGGTGCCATA-3′) were used to amplify the bla _CTX-M gene (Mollenkopf et al., 2012). bla _CTX-M genes were bidirectionally Sanger sequenced using the corresponding PCR amplification primers and analyzed using the Basic Local Alignment Search Tool (BLAST; http://blast.ncbi.nlm.nih.gov).

For culture of Salmonella spp., the second gauze sponge was incubated overnight in 36 mL of tetrathionate broth at 37°C. The following day, a 100-μL aliquot was added to 10 mL of Rappaport–Vassiliadis broth, and the samples were incubated for 24 h at 42°C. Using a sterile swab, the samples were inoculated onto Xylose Lactose Tergitol 4 agar and streaked for isolation, then incubated overnight at 37°C. The plates were checked for growth, and a single isolated black colony from each positive growth was transferred to MacConkey agar and incubated overnight at 37°C. Lactose-negative suspect Salmonella colonies were confirmed by biochemical tests, including triple sugar iron agar, urea broth, and serogrouping by agglutination (Tille, 2014).

Retail packages of ground chicken were purchased on each sampling date on-site at a retail store adjacent to the processing plant and screened for the presence of both Salmonella and Enterobacteriaceae resistant to extended-spectrum cephalosporins. Triplicate 4-g aliquots from each ground meat package were processed as described above for broiler cage swabs. Extended-spectrum cephalosporin resistance genes were confirmed by conventional PCR, as described above. Because the retail ground chicken was purchased on the same day as the transport cages were sampled, they are not intended to represent the product from the same broilers present in transport cages on the day of cage sampling. They only provide information about the similarity of isolates recovered from the two sources.

To examine the genetic similarity of all Salmonella isolates recovered from cage swabs and meat packages, pulsed-field gel electrophoresis (PFGE) genotyping (CHEF-DRIII; Bio-Rad Laboratories, Hercules, CA) was performed on total genomic DNA. Agarose plugs prepared from Salmonella isolates were digested using XbaI (Promega, Madison, WI) following previously reported protocols (Ribot et al., 2006). After electrophoresis, banding patterns were compared and levels of similarity assigned using generally accepted criteria (Tenover et al., 1995). Salmonella isolates were compiled into pulsotype groups by using the Dice coefficient similarity index and the unweighted pair-group method with arithmetic mean (UPGMA) with clustering settings of 1.00% optimization and 1.00% band position tolerance using Bionumerics software (Applied Maths, Kortrijik, Belgium). A representative isolate from each pulsotype was sent to NVSL for serotyping.

Results

We collected a total of 425 individual cage swab samples from 13 transport trailers over a 9-week period. We collected an average of 32.7 cage swabs per trailer, with a range of 5–71 samples per trailer based on their availability for sampling. We recovered Salmonella spp. from 111 (26.1%) samples, with the positive proportion ranging from 0% to 56% of cage samples on individual sampling dates (Table 1). Salmonella recovery by trailer ranged from 0% (0/39) to 60% (3/5) (Table 2).

Table 1.

Frequency of Recovery of Salmonella spp. and Escherichia coli Expressing the AmpC or Extended-Spectrum Beta-Lactamase Resistance Phenotypes from Broiler Transport Cage Swabs by Sampling Date

				E. coli
		Salmonella spp.		AmpC phenotype ^a		ESBL phenotype ^b
Sampling date	Samples (n)	Isolates	%	Isolates	%	Isolates	%
May 13	25	11	44	24	96	3	12
May 20	50	0	—	49	98	5	10
June 12	50	28	56	41	82	33	66
June 17	50	7	14	43	86	9	18
June 24	50	6	12	48	96	5	10
July 8	50	20	40	49	98	3	6
July 15	50	15	30	48	96	4	8
July 22	50	11	22	41	82	17	34
July 29	50	13	26	46	92	15	30
Total	425	111	26.1	389	91.5	94	22.1

The AmpC phenotype is defined as growth in the presence of 8 μg/mL cefoxitin.

The ESBL phenotype is defined as growth in the presence of 4 μg/mL cefepime.

ESBL, extended-spectrum beta-lactamase.

Table 2.

Frequency of Recovery of Salmonella spp. and Escherichia coli Expressing the AmpC or Extended-Spectrum Beta-Lactamase Resistance Phenotypes from Broiler Transport Cage Swabs by Trailer

				E. coli
		Salmonella spp.		AmpC phenotype ^a		ESBL phenotype ^b
Trailer No.	Samples (n)	Isolates	%	Isolates	%	Isolates	%
13	39	0		36	92	6	15
17	20	4	20	18	90	0
34	55	14	25	53	96	12	22
36	6	2	33	3	50	1	17
46	47	20	43	45	96	11	23
47	31	9	29	31	100	7	23
51	25	4	16	23	92	3	12
56	71	24	34	63	89	22	31
63	52	12	23	50	96	10	19
64	54	13	24	47	87	11	20
67	5	3	60	5	100	4	80
69	10	4	40	7	70	4	40
83	10	2	20	8	80	3	30
Total	425	111	26	389	92	94	22

The AmpC phenotype is defined as growth in the presence of 8 μg/mL cefoxitin.

The ESBL phenotype is defined as growth in the presence of 4 μg/mL cefepime.

The 111 Salmonella spp. isolates recovered from trailer swabs primarily represented 4 serotypes, Enteritidis (n = 34), Kentucky (n = 26), Mbandaka (n = 6), and Rough O:g,m:- (n = 40). Within serotypes, PFGE analysis indicated that the Salmonella Kentucky, Salmonella Mbandaka, and Salmonella Rough O:g,m:- each represented single highly related strains. Within the Salmonella Enteritidis serotype, we identified two distinct strains (strain C.1 n = 20; strain C.2 n = 14) based on clustering of pulsotypes. Five additional isolates representing diverse pulsotypes were not serotyped.

The distribution of Salmonella serovars by sampling date is summarized in Figure 1. Salmonella Enteritidis isolates were recovered on 7 different sampling dates and from 10 different trailers. One of the Salmonella Enteritidis strains (n = 20) was recovered on 6 different weeks and from seven different trailers. The second Salmonella Enteritidis strain (n = 14) was recovered on five different weeks and from seven different trailers. Salmonella Kentucky isolates were recovered on three different sampling dates and from eight different trailers. Salmonella Mbandaka isolates were recovered on two different dates and from four different trailers. Five of the Salmonella Mbandaka isolates were recovered on the same date (week 1) from three different trailers, and the one additional isolate was recovered on week 7 from a fourth trailer. Rough O:g,m:- isolates were recovered on six different dates and from eight different trailers. All four serotypes were recovered at least once from three trailers, and multiple serotypes were recovered from nine trailers.

FIG. 1.

Distribution of Salmonella spp. serovars recovered from broiler transport cage swabs by sampling date.

We recovered E. coli expressing an AmpC beta-lactamase resistance phenotype from 389 (91.5%) transport cage swabs (Table 1). The proportion of cage swab samples with isolates expressing the AmpC resistance phenotype ranged from 82% to 98% on individual sampling dates. The proportion of cage swab samples that produced isolates expressing an AmpC beta-lactamase phenotype ranged from 50% to 100% for individual trailers (Table 2). Sixty-four (91.4%) of a subset of 70 E. coli expressing the AmpC beta-lactamase phenotype were confirmed to harbor bla _CMY by conventional PCR.

We recovered E. coli expressing an ESBL resistance phenotype from 94 (22.1%) transport cage swab samples (Table 1), ranging from 6% to 66% on individual sampling dates. The proportion of cage swab samples expressing an ESBL resistance phenotype ranged from 0% to 80% for individual trailers (Table 2). Eighty-seven (98.8%) of a subset of 88 E. coli expressing the ESBL phenotype were confirmed to harbor bla _CTX-M group 1 alleles by PCR.

A total of 72 ground chicken aliquots from 24 individual retail packages were screened. Salmonella were recovered from two (2.8%) retail ground chicken samples purchased on two different sampling dates. One of the isolates recovered from ground chicken was Salmonella Enteritidis (strain 1) that was highly related on PFGE analysis to isolates recovered from cage swabs collected from two different trailers on the same day. The second isolate recovered from ground chicken was another Salmonella Enteritidis pulsotype (strain 2) that was highly related to an isolate recovered from a trailer the same day and a second trailer the previous week.

E. coli expressing an AmpC beta-lactamase resistance phenotype were recovered from 56 (77.8%) retail ground chicken samples. E. coli expressing an ESBL resistance phenotype were recovered from eight (11.1%) retail ground chicken samples. We did not recover isolates resistant to meropenem from cage swab or ground meat samples.

Discussion

We observed that in this production system, Salmonella spp. frequently contaminated the broiler transport cages that were routinely used to move broiler chickens from grower houses to the processing plant. Over 25% of 425 transport cage swabs collected from 13 trailers harbored Salmonella isolates. This finding is not surprising given the frequency that broilers are colonized and shed Salmonella. Environmental samples from broiler houses indicate that broiler chicken populations are colonized by Salmonella at rates approaching 100% (Berghaus et al., 2013). Salmonella fecal shedding by broilers during transport to slaughter would be expected to contaminate transport cages and is likely the source of the Salmonella isolates we recovered with cage swabs. Without adequate cleaning and disinfection, transport cages could serve as a potential source for exposure of uncolonized broilers just before processing for introduction into the food supply, as has been previously suggested (Heyndrickx et al., 2002).

While we frequently recovered Salmonella isolates from transport cage swabs, those isolates represented relatively few Salmonella strains. Five unique strains were recovered repeatedly over time and from cages on multiple transport trailers over the course of the study. This result suggests that Salmonella strains can persist over time in broiler populations within a production system. Salmonella strains that persist in broiler production systems have been previously reported (Lahellec et al., 1986; Liljebjelke et al., 2005; Alali et al., 2010) and may be, in part, the result of specific broiler production practices. This observation also suggests that Salmonella strains may persist in broiler transport cages where they could serve as one of the potential sources for exposure, contamination, or infection of broilers just before processing for entry into the food supply as fresh retail meat.

Exposure to Salmonella just before slaughter has been shown to increase the risk of Salmonella infection and shedding in swine (Hurd et al., 2001). Similar to what we observed with broiler transport cages, swine lairage facilities are frequently contaminated with Salmonella (Swanenburg et al., 2001; Hurd et al., 2002), and the same Salmonella strains recovered from lairage facilities have been recovered from carcasses of swine (De Busser et al., 2011). Swine at processing facilities can quickly become infected with Salmonella during holding in lairage, with holding times as short as 2 h associated with increased risk (Swanenburg et al., 2001; Hurd et al., 2002). While broilers are not unloaded into lairage at processing before slaughter, Salmonella contamination of broiler transport cages may provide a similar opportunity for uncolonized broilers to be exposed during transport to processing. This might increase the risk of infection and shedding in broilers just before their entry into the processing plant, which could then increase food safety risk (Heyndrickx et al., 2002).

We recovered Salmonella isolates from two packages of ground chicken purchased at the processing facility retail store. This result likely underestimates the true prevalence of Salmonella contamination of ground chicken from this facility because our Salmonella culture protocol utilizing 4 g of sample is expected to have lower sensitivity than protocols recommended for Hazard Analysis and Critical Control Point (HACCP) monitoring by the USDA FSIS that utilize 25–325 g of sample. Both of the Salmonella strains we recovered from the fresh, retail meat products that we purchased at the processing facility on-site retail store were strains that we also found in the transport cages. Salmonella frequently contaminate poultry carcasses during processing and fresh, retail poultry products (Liljebjelke et al., 2005; Volkova et al., 2010; Mollenkopf et al., 2014), so it is not surprising that we found the same strains contaminating both the transport cages and fresh meat products produced at the same facility. Previously, Salmonella-negative broilers have been shown to shed Salmonella after transport (Marin and Lainez, 2009). If previously noncolonized broilers are exposed to Salmonella during transport in contaminated cages and enter the processing facility internally or externally carrying this pathogen, then the potential for the resulting fresh meat products to be similarly contaminated may increase, but this relationship has not been established.

E. coli expressing AmpC and ESBL phenotypes were frequently recovered from the transport cage swabs and from fresh, retail poultry products. Contamination of fresh, retail chicken meat with extended-spectrum cephalosporin-resistant E. coli has been previously reported (Mollenkopf et al., 2014). Similar to Salmonella, extended-spectrum cephalosporin-resistant E. coli contamination of transport cages likely reflects shedding of these organisms by broilers during transport. However, these multiresistant E. coli strains may also pose a risk of infection and shedding to uninfected broilers subsequently transported in the same cages to the processing facility.

Our results suggest that appropriate cleaning and disinfection of broiler transport cages should be considered as possible interventions to help reduce broiler exposure to Salmonella and ESC-resistant E. coli contamination just before their entry into the food supply as fresh, retail poultry products. Cleaning and disinfection have been shown to reduce Salmonella contamination of the lairage at swine facilities (Schmidt et al., 2004; Boughton et al., 2007). However, similar data for broiler transport cages have not been reported, and additional research is needed.

Footnotes

Disclosure Statement

No competing financial interests exist.

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