Ceftiofur Use in Finishing Swine Barns and the Recovery of Fecal Escherichia coli or Salmonella spp. Resistant to Ceftriaxone

Abstract

The objective of this study was to investigate the association between ceftiofur use policy in finishing swine barns and recovery of fecal Escherichia coli or Salmonella spp. resistant to ceftriaxone. The study population included 54 finishing swine barns from three companies located in North Carolina. The barns were each classified according to their reported therapeutic ceftiofur use rates of “Rare,” “Moderate,” and “Common.” Fecal samples from the barns were cultured for the presence of E. coli and Salmonella spp. resistant to ceftriaxone using selective media designed to recover rare organisms expressing the AmpC β-lactamase phenotype. A total of 1899 swine fecal samples yielded 1193 E. coli (63%) resistant to ceftriaxone. Recovery rates by ceftiofur use classification were 45% for Rare, 73% for Moderate, and 68% Common ceftiofur use groups. Barns reporting Rare ceftiofur use had a lower odds of recovery of E. coli (OR=0.32; p<0.001) resistant to ceftriaxone compared to Common use barns. The overall Salmonella spp. prevalence was 63.8% (n=714). Of these, 65 Salmonella were resistant to ceftriaxone with the highest rate (6%) found in the Common ceftiofur use group, followed by Rare (4.1%) and Moderate (0.15%). The odds of recovery of Salmonella resistant to ceftriaxone were similar for barns with ceftiofur use classified as Rare and Common. Samples from barns with ceftiofur use classified as Moderate had a lower odds (OR=0.02; p<0.01) of recovery of Salmonella resistant to ceftriaxone than barns classified as Common. Our result is consistent with the hypothesis that the use of ceftiofur in finishing swine barns, beyond its rare application, may influence the recovery of enteric E. coli with resistance to cephalosporin drugs, although other unmeasured factors appear to be important in the recovery of cephalosporin-resistant Salmonella. The dissemination of enteric bacteria with resistance to cephalosporins has the potential to impact both veterinary and human therapeutic treatment options.

Introduction

I n food animal production operations, antimicrobial drug use for therapeutics, prophylactic treatment, and growth promotion is an integral tool used industry wide. While there are many antimicrobials used in agriculture with no human therapeutic derivative (bacitracin, bambermycins, carbadox, and ionophores), there are many others that are closely related to human antibiotics, including the β-lactam penicillins and cephalosporins, sulphonamides, tetracyclines, macrolides, lincosamides, streptogramins, and quinolones (Phillips et al., 2004). One example is ceftiofur, the only third-generation cephalosporin labeled for veterinary use. This antimicrobial is effective against Gram-negative bacteria and is commonly applied in swine production operations as an injectable therapeutic (USDA, 2007). Ceftriaxone, a third-generation cephalosporin similar to ceftiofur, is the human-medicine drug of choice for treatment of invasive Gram-negative bacterial infections in children (e.g., Salmonella spp. and Shigella spp.), with over 16 million prescriptions written between 2000 and 2005 (Powers, 2010). Both the World Health Organization (WHO) and the U.S. Food and Drug Administration (US-FDA) have recognized third-generation cephalosporins, like ceftriaxone, as critically important to human medicine (WHO, 2007; FDA, 2009).

Resistance of Gram-negative enterics such as Salmonella spp. and Escherichia coli to third-generation cephalosporins is a major concern for both veterinary and human medicine. Resistance to these drugs occurs through a natural selection process of genetically mediated survivability in the presence of antibiotics. β-lactam antibiotics such as penicillins and cephalosporins share a common four-atom cyclic amide ring (lactam ring). This ring provides competitive inhibition of bacterial enzymes required for cell wall synthesis, resulting in ceased cell division and cell lysis (Rupp and Fey, 2003). Through this mechanism, exposure of susceptible bacteria to β-lactam drugs ultimately results in cell death.

β-lactamase enzymes can be produced by many bacteria, including both Salmonella spp. and E. coli. These enzymes disable β-lactam antimicrobials by breaking open the amide ring. Hence, the ability of an organism to produce a β-lactamase can confer bacterial resistance to β-lactam drugs. Selective pressure on bacterial populations when β-lactams are present may select for the widely disseminated AmpC β-lactamase gene, bla _CMY, which is typically located on a mobile plasmid (Tragesser et al., 2006; Heider et al., 2009). E. coli, being closely related to other pathogenic enteric bacteria, for example, Salmonella, is reported to act as a reservoir of AmpC bla _CMY genes available to other bacteria for further spread of β-lactam resistance (Winokur et al., 2000).

The association between ceftiofur use in swine and the emergence of resistant enteric flora is not known, and the potential animal and public health implications have not been evaluated. Associations between ceftiofur use and resistance of enteric flora in dairy and beef cattle have been previously reported (Jiang et al., 2006; Lowrance et al., 2007), suggesting the possibility of a similar relationship in other intensively managed livestock populations. In this cross-sectional study, we hypothesize that recovery of Salmonella spp. and E. coli with resistance to ceftriaxone from swine fecal samples may increase with increased ceftiofur use.

Materials and Methods

From three large North Carolina commercial swine production companies, 54 distinct finishing barns were recruited for voluntary participation in this study. These barns were similar in size and design, and each housed approximately 960 pigs. Therapeutic ceftiofur use in each barn was classified as “Rare,” “Moderate,” or “Common” (n=18 barns for each) based on frequency of ceftiofur use reported by the veterinarians responsible for the health of the animals on these farms. Barns with ceftiofur use classified as Rare did not include ceftiofur in standard treatment protocols for common health problems such as respiratory disease, and it would be unusual for any of these pigs to receive therapy with ceftiofur. Barns with ceftiofur use classified as Moderate included ceftiofur as one of the therapeutic options in standard treatment protocols, although not as the first choice, and it would not be unusual for these pigs to receive therapy with ceftiofur although other drugs would be used more frequently. Barns with ceftiofur use classified as Common included ceftiofur as the therapy of choice in standard treatment protocols for common health conditions, and ceftiofur was the most frequently used therapy. Ceftiofur use was not directly measured in these barns beyond these general ceftiofur use policies.

From each of the barns, fresh fecal samples were systematically collected from the floor of occupied pens during a single visit to each barn. A target number of 36 individual samples per barn was identified, but was proportionally reduced when not all pens in the barns were full. To avoid cross-contamination, each sample was collected using a new latex glove and sterile plastic centrifuge tube. On the day of collection, samples were transported by overnight courier to our laboratory at The Ohio State University for processing the day following field collection.

To recover E. coli expressing the characteristic AmpC β-lactamase phenotype from the fecal samples, 4-g aliquots were first incubated in 36 mL of nutrient broth containing cefoxitin 4 μg/mL overnight at 37°C, and then aseptically inoculated and streaked for isolation onto MacConkey agar with ceftriaxone 8 μg/mL with overnight incubation at 37°C. Characteristic lactose-fermenting colonies that were subsequently indole positive were presumed to be E. coli. We have previously used this procedure to successfully recover fecal E. coli carrying bla _CMY (Tragesser et al., 2006; Heider et al., 2009).

From all ceftriaxone-resistant E. coli we randomly selected 3 isolates from each of the 54 barns, minus 3 isolates from barns that yielded less than 3 total isolates for a subset of 159 E. coli isolates. Representative isolated colonies were evaluated using standard PCR procedures for confirmation of both bacterium and the presence of the AmpC β-lactamase gene, bla _CMY (Winokur et al., 2000; Bayardelle and Zafarullah, 2002).

For the culture of Salmonella spp., 10-g fecal aliquots were homogenized with 90 mL of tetrathionate broth (TTB) containing iodine and incubated overnight at 37°C. The following day, 0.10 mL of TTB was pippetted to 10 mL of Rappaport-Vassiliadis R10 Broth (RV) and incubated at 42°C overnight. On day 3, the RV was inoculated to Xylose-Lysine-Tergitol 4 agar (XLT-4) and again incubated overnight at 37°C. The XLT-4 plates were then evaluated for the presence of characteristic black colonies. An isolated colony from each presumptive positive sample was streaked for isolation on MacConkey agar and again incubated overnight at 37°C. Non-lactose fermenting organisms were confirmed as Salmonella using standard biochemical reactions, including triple sugar iron agar, urea broth, and polyvalent antisera testing.

To identify Salmonella expressing the bla _CMY phenotype, all isolates were incubated overnight at 37°C on MacConkey agar containing ceftriaxone 8 μg/mL, with surviving isolates transferred to MacConkey agar containing cefoxitin 4 μg/mL and again incubated overnight at 37°C. Salmonella isolates with reduced susceptibility to both the second- and third-generation cephalosporins were tested by PCR to confirm the presence of bla _CMY (Winokur et al., 2000).

From the E. coli and Salmonella spp. isolates confirmed to contain bla _CMY, 16 E. coli and 25 Salmonella representative isolates were selected for evaluation of minimum inhibitory concentrations (MICs) to 26 antimicrobial drugs (Table 1). Using E. coli ATCC 25922, Pseudomonas aeruginosa ATCC 27852, and Enterococcus faecalis ATCC 29212 as quality control organisms, we evaluated the MICs for the 41 isolates using a semi-automated broth microdilution system (Sensititre Sensitouch; TREK Diagnostic Systems, Cleveland, OH) following manufacturer's and Clinical and Laboratory Standards Institute guidelines (CLSI, 2008).

Table 1.

Minimum Inhibitory Concentrations to 26 Antimicrobial Drugs for 16 Escherichia coli and 25 Salmonella spp. Isolates Containing bla _CMY Recovered from Finishing Swine Fecal Samples

Thicker lines represent susceptible breakpoints; thinner lines represent resistant breakpoints when available.

Total of 24 E. coli isolates reported for pipercillin/tazobactam. Result from one E. coli isolate was invalid.

All statistical modeling was performed using SAS, Version 9.1 (SAS Institute, Inc., Cary, NC). The outcome of interest was the recovery of E. coli or Salmonella spp. resistant to ceftriaxone from individual fecal samples. Logistic regression was used to compare the odds of recovery of E. coli or Salmonella spp. resistant to ceftriaxone across the three ceftiofur use-level groups: Rare, Moderate, and Common. Generalized estimating equations were used to account for clustering of samples within barns.

Results

From the 54 barns included in the study, 579 fresh fecal samples were collected from those categorized with Rare ceftiofur use, 648 samples from Moderate, and 672 from Common totaling 1899 fresh fecal samples. Of these total fecal samples collected, 1193 were found to contain E. coli resistant to ceftriaxone—a 63% recovery rate across all three ceftiofur usage categories. Additionally, E. coli resistant to ceftriaxone recovered from the Rare ceftiofur use barns was 45%, Moderate was 73%, and 68% from Common. From the subset of 159 ceftriaxone-resistant E. coli isolates, PCR results indicated that 138 isolates (86.8%) contained bla _CMY.

Salmonella spp. were also recovered from 714 (37.6%) of the 1899 fecal samples. Of these, 65 (9.1% of isolates, 3.4% of samples) were resistant to ceftriaxone and were confirmed by PCR to contain bla _CMY. Recovery rates of Salmonella spp. containing bla _CMY by ceftiofur use classification were 4.1% from barns classified as Rare ceftiofur use, 0.15% for barns classified as Moderate, and 6.0% as Common ceftiofur use.

Results of logistic regression models with generalized estimating equations found that samples from barns where ceftiofur use was classified as Rare had a lower odds of recovery of E. coli (OR=0.32; p<0.001) resistant to ceftriaxone than samples from barns where ceftiofur use was classified as Common (Table 2). The odds of recovery of E. coli resistant to ceftriaxone were similar for samples from barns classified as Moderate and Common ceftiofur use (Table 2). In contrast, the odds of recovery of Salmonella resistant to ceftriaxone were similar for barns with ceftiofur use classified as Rare and Common. Samples from barns with ceftiofur use classified as Moderate had a lower odds (OR=0.02; p<0.01) of recovery of Salmonella resistant to ceftriaxone than barns classified as Common (Table 2).

Table 2.

Results of Logistic Regression Models with Generalized Estimating Equations for Predicting the Effect of Rare, Moderate, or Common Ceftiofur Use in Finishing Swine Barns on the Odds of Recovery of Ceftriaxone-Resistant Escherichia coli or Salmonella spp. from Fecal Samples

Model	Ceftiofur use	OR	95% CI (OR)	p-Value	LS mean
Escherichia coli
	Rare	0.32	0.17 to 0.63	0.001	40.3%
	Moderate	1.40	0.74 to 2.7	0.31	74.9%
	Common	1.0	—	—	68.0%
Salmonella spp.
	Rare	0.68	0.22 to 2.2	0.51	4.15%
	Moderate	0.02	0.003 to 0.18	<0.001	0.15%
	Common	1.0	—	—	5.95%

LS, least squares.

Discussion

We found that the odds of recovery of ceftriaxone-resistant E. coli were higher in fecal samples from barns where ceftiofur use was common than in fecal samples from barns where ceftiofur use was rare. The odds of recovery of these isolates were similar for fecal samples from barns with ceftiofur use classified as either Moderate or Common. This result suggests that any ceftiofur use in finishing swine barns other than its rare application may provide adequate selection pressure to allow the widespread dissemination of bla _CMY in the enteric flora of the pigs in the barn.

We found that the odds of recovery of ceftriaxone-resistant Salmonella were lowest in fecal samples from barns where ceftiofur use was reported to be Moderate. The odds of recovery of these resistant isolates were similar for fecal samples from barns with ceftiofur use classified as either Rare or Common. This result suggests that other unmeasured management or environmental factors are more important than ceftiofur use in determining the presence of ceftriaxone-resistant Salmonella. Our conflicting results for the association of ceftiofur use with the recovery of resistant E. coli versus Salmonella spp. in swine finishing barns may reflect the role of commensal E. coli as a reservoir of bla _CMY for other organisms or potential pathogens such as Salmonella spp. (Winokur et al., 2001; Lefebvre et al., 2008).

Ceftiofur is commonly used for the therapeutic treatment of clinical disease in swine operations, with 42.1% of grower/finisher operations reporting its use, primarily for the treatment of respiratory disease (USDA, 2007). However, estimates of individual pig treatment rates with ceftiofur in finishing barns are not available. Our observation that moderate and common use of ceftiofur result in similar recovery rates of E. coli resistant to ceftriaxone suggests that there is a critical ceftiofur use baseline between rare and moderate use above which bla _CMY will disseminate in the population. Our results are not consistent with a linear dose–response relationship between ceftiofur use and the recovery of bla _CMY in finishing swine barns.

A similar association between ceftiofur use in dairy cattle and recovery of enteric E. coli resistant to ceftriaxone has been investigated, with inconsistent results. One study of Ohio dairy herds found that ceftiofur use was associated with increased odds of recovery of E. coli with reduced susceptibility to ceftriaxone at the herd level, but not at the individual cow level (Tragesser et al., 2006). They hypothesized that once ceftiofur was used in a dairy herd at any level, bacterial resistance genes were rapidly disseminated among all individuals in the herd. Our results are consistent with this hypothesis in that any use of ceftiofur in swine finishing barns beyond its rare application resulted in similar recovery rates of ceftriaxone-resistant E. coli.

A more recent study of Ohio dairy herds with a high frequency of ceftiofur use reported no association between the proportion of cows treated with ceftiofur and reduced ceftriaxone susceptibility of enteric E. coli and Salmonella (Heider et al., 2009). Another recent study evaluated the effects of ceftiofur use on the antibiotic susceptibility profile of enteric E. coli in dairy cattle and found that antibiotic treatment was not linked to amplification or emergence of resistant E. coli (Singer et al., 2008), but instead reduced the susceptible bacterial populations in the flora to a level that allowed rare resistant populations to be detected. Others have reported similar results investigating ceftiofur and its role in dissemination of cephalosporin resistance in commensal E. coli isolates from cattle (Alali et al., 2009; Daniels et al., 2009; Mann et al., 2011). Taken together, the results of these studies in dairy cattle support a possible association between ceftiofur use and either the enhanced detection or dissemination of enteric bacteria with reduced-susceptibility to ceftriaxone in cattle operations. However, differences in the therapeutic and prophylactic use of ceftiofur in the swine industry may result in a different impact of ceftiofur use in swine production.

In finishing swine operations, the frequency of ceftiofur use is generally not as high as in dairy cattle populations (USDA, 2007, 2008). However, our results suggest a similar relationship between ceftiofur use and the recovery of E. coli resistant to ceftriaxone. Similar results have been reported in Europe, where the prophylactic use of ceftiofur in day old piglets was associated with the recovery of E. coli with reduced susceptibility to cefotaxime (Jørgensen et al., 2007). In China, a survey of two large commercial swine farms revealed an increase in the prevalence of β-lactamase producing E. coli isolates over a 5-year period following the approval of ceftiofur for veterinary use (Tian et al., 2009). However, the true impact of ceftiofur use in swine on the dissemination of resistant organisms has not been well characterized. Our results are consistent with the hypothesis that any regular use of ceftiofur in finishing swine barns influences the frequency of enteric E. coli resistant to cephalosporin drugs, although other factors appear to be important in the dissemination of cephalosporin-resistant Salmonella.

While ceftiofur use has been previously associated with extended-spectrum β-lactamase resistance in individual animals (Jiang et al., 2006; Jørgensen et al., 2007) and in livestock populations (Tragesser et al., 2006; Lowrance et al., 2007), other factors might also play an important role in the dissemination of β-lactamase genes in the flora of livestock. For example, bla _CMY is commonly located on a large plasmid that can carry additional resistance genes (Winokur et al., 2000; Rankin et al., 2002). As a result, the selection pressure provided by the common use of non-β-lactam antimicrobial drugs such as tetracycline could lead to the dissemination of β-lactamase genes. It has been reported that Gram-negative isolates from fecal flora of pigs exposed to subtherapeutic chlortetracycline in feed were more likely to be resistant to ceftriaxone than isolates from untreated control pigs (Funk et al., 2006). However, others have reported that exposure to tetracycline and other non-β-lactam antimicrobial drugs was not associated with the frequency of ceftiofur resistance in the flora of pigs (Wagner et al., 2008), cattle (Alexander et al., 2008; 2009), and chickens (Bonnet et al., 2009). In addition, the possible role of coselection in the dissemination of bla _CMY and other β-lactamase resistance genes in bacterial flora has not been reported. It is possible that coselection by other antimicrobial drugs may have impacted our observed results, although our study was not designed to address that hypothesis. Future studies should attempt to account for the possible role of coselection when investigating the relationship between extended-spectrum cephalosporin use and resistance in livestock populations.

It is important to note that we examined the basic relationship between ceftiofur use policy and the recovery of resistant enteric bacteria without consideration of the complex management, environment, and animal factors that can influence the development and dissemination of antimicrobial resistance in livestock populations. The inconsistent association we observed between reported ceftiofur use and the recovery of ceftriaxone-resistant E. coli and Salmonella spp. is also likely indicative of the complex relationship between veterinary antimicrobial use and resistance. As a result, we cannot draw strong conclusions regarding this putative relationship. However, our results do support the need for a better understanding of these complex relationships.

Conclusions

Limitations of our study design prevent us from drawing strong conclusions, but our result is consistent with the hypothesis that the use of ceftiofur in finishing swine barns, beyond its rare application, may influence the recovery of enteric E. coli with resistance to cephalosporin drugs, although other unmeasured factors appear to be important in the recovery of cephalosporin-resistant Salmonella. These conflicting results for the association of ceftiofur use with the recovery of resistant E. coli versus Salmonella spp. in swine finishing barns may reflect the role of commensal E. coli as a reservoir of bla _CMY for potential pathogens such as Salmonella spp. The dissemination of enteric bacteria with resistance to cephalosporins has the potential to negatively impact both veterinary and human therapeutic treatment options.

Footnotes

Acknowledgment

This research was supported by the USDA CSREES IREE NIFSI.

Disclosure Statement

No competing financial interests exist.

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