Antimicrobials are critical to treat, control, and prevent disease in swine and other food animals. Responsible and timely antibiotic intervention is vital in controlling and mitigating disease incidence and spread, such as in swine respiratory disease (SRD) complex, which can endanger herd health and a sustainable food supply resulting in economic and commercial loss.¹ Of all the diseases that affect growing and finishing pigs, SRD is the most economically important as it is highly prevalent among indoor production facilities and can be difficult to treat and control. The treatment and control of SRD requires an understanding of the complexities and interaction between the organisms that are present as well as management of the environment in which the pigs are raised.² Primary pathogens for SRD complex may include Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, and Bordetella bronchiseptica, as well as viral agents. Common secondary pathogens include Pasteurella multocida, Streptococcus suis, Glaesserella parasuis, Actinobacillus suis, and Salmonella Choleraesuis. These primary and secondary multi-etiologic pathogens act together to increase the severity and duration of SRD.³

Antimicrobial surveillance among veterinary bacterial pathogens obtained from clinical specimens provides a platform from which to detect emergence of resistance in animal populations. While veterinary diagnostic laboratories throughout North America and Europe provide important antimicrobial susceptibility information for clinical isolates submitted by the attending veterinarian or animal caretaker, the susceptibility results are not typically examined. Few surveillance programs monitor susceptibility in swine pathogens nationally or internationally.^4-6 Portis et al⁴ reported minimal inhibitory concentration (MIC) values for 7 antimicrobials against A pleuropneumoniae, P multocida, and S suis isolated from diseased swine in the United States and Canada over a 10-year period (2001-2010) and concluded that most isolates showed high rates of susceptibility to all antimicrobials tested. Additionally, Sweeney et al⁵ reported MIC values for 10 antimicrobials against A pleuropneumoniae, B bronchiseptica, P multocida, and S suis isolated from diseased swine in the United States and Canada over a 5-year period (2011-2015) and concluded that most isolates showed high rates of susceptibility to all antimicrobials tested except tetracycline.

Continuing this surveillance program, we report the percentages of A pleuropneumoniae, B bronchiseptica, P multocida, and S suis pathogens isolated from swine in the United States and Canada that were susceptible to the veterinary antimicrobials ampicillin, ceftiofur, danofloxacin, enrofloxacin, florfenicol, penicillin, tetracycline, tilmicosin, trimethoprim-sulfamethoxazole (TMP-SMX), and tulathromycin. This paper presents the findings of the most contemporaneous 5-year surveillance period on SRD pathogens collected in North America from 2016 to 2020.

Animal care and use

Diagnostic submission data from clinical submissions were used in this study, therefore no animal use protocol was required.

Materials and methods

Laboratory participants and isolate characterization

Veterinary diagnostic laboratories from the United States and Canada participated in this surveillance study. The regions from which isolates were obtained are shown in Table 1.

All A pleuropneumoniae, B bronchiseptica, P multocida, and S suis isolates were recovered from diseased or dead pigs. Laboratories selected isolates based on their own protocols and were requested not to use antimicrobial susceptibility as a criterion for selection. Laboratories were also requested to submit no more than eight isolates per quarter year to prevent over-representation from any one geographic area. Each participating laboratory was also requested to send no more than one isolate of each bacterial species from a herd each quarter year to prevent the over-representation of bacterial clones from one region.^4,5

Bacterial isolates were identified to the species level by each participating laboratory before shipment to a central laboratory for susceptibility testing and the species identifications were confirmed at Zoetis (Kalamazoo, Michigan) using Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS; Bruker). All isolates were stored in approximately 1.0 mL trypticase soy broth (BD Biosciences) supplemented with 10% glycerol and stored at approximately -70°C until tested.

Determination of MIC values

In vitro susceptibility data were generated annually by performing MIC testing at a central laboratory (Microbial Research Inc) and followed Clinical and Laboratory Standards Institute (CLSI) standardized methods and quality control guidelines during susceptibility testing.⁷ The MIC values for all isolates were determined using a dehydrated broth microdilution system (Sensititre System; Thermo Fisher Scientific) which conforms to CLSI standards for testing of veterinary pathogens.⁷ Additionally, the central laboratory followed all manufacturer instructions for quality assurance and quality control when using the Sensititre plates. Direct colony suspensions were used and prepared at a final bacterial concentration of approximately 5 × 10⁵ colony forming units/mL. Custom-made 96-well microtiter panels included serial doubling dilutions of the antimicrobials ampicillin, ceftiofur, danofloxacin, enrofloxacin, florfenicol, penicillin, tetracycline, tilmicosin, TMP-SMX, and tulathromycin. All concentration ranges for antimicrobials were chosen to encompass appropriate quality control ranges and published clinical breakpoints, and appropriate quality-control organisms were included with each testing date.⁸

Results

Quality control

The quality control organisms used in this study included Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 29213, Streptococcus pneumoniae ATCC 49619, and A pleuropneumoniae ATCC 27090. Although not shown for this study, MIC values for all appropriate quality control organisms were acceptable when all study isolates were tested against antimicrobials on each date of testing.

A pleuropneumoniae

The MIC distributions, MIC₅₀ values, and MIC₉₀ values for 10 antimicrobials tested against A pleuropneumoniae (n = 250) are reported in Table 2. The CLSI has established clinical breakpoints for A pleuropneumoniae against ampicillin, ceftiofur, enrofloxacin, florfenicol, tetracycline, tilmicosin, and tulathromycin. Actinobacillus pleuropneumoniae susceptibility to ampicillin increased overall from 85.7% in 2016 (susceptible breakpoint ≤ 0.5 µg/mL) to 97.6% in 2020, but decreased to 83% in 2018. The percentage of isolates susceptible to ceftiofur over the 5-year study period was 100% (susceptible breakpoint ≤ 2 µg/mL) and the MIC₉₀ values were ≤ 0.03 µg/mL. The percentage of susceptibility to enrofloxacin was very high (100% in 2016 and 2018-2020; breakpoint ≤ 0.25 µg/mL), and the MIC₉₀ values over the study period were 0.06 to 1 µg/mL; florfenicol was 100% susceptible (breakpoint ≤ 2 µg/mL), with MIC₉₀ values at 0.5 µg/mL. Actinobacillus pleuropneumoniae susceptibility to tetracycline (breakpoint ≤ 0.5 µg/mL) was very low, with a susceptibility range of 0% to 10.6%, while tilmicosin susceptibility (breakpoint ≤ 16 µg/mL) ranged from 96.8% in 2016 to 100% in 2020. There was 100% percent susceptibility of A pleuropneumoniae to tulathromycin (breakpoint ≤ 64 µg/mL) and MIC₉₀ values ranged from 32 to 64 µg/mL. While CLSI-approved susceptible breakpoints have not been established for danofloxacin, penicillin, or TMP-SMX, the MIC₉₀ values were determined as 0.06 to 1 µg/mL, 0.5 to ≥ 32 µg/mL, and ≤ 0.06 to 0.12 µg/mL, respectively, from 2016 to 2020.

B bronchiseptica

The MIC distributions, MIC₅₀ values, and MIC₉₀ values for 10 antimicrobials tested against B bronchiseptica (n = 602) are reported in Table 3. The CLSI has established clinical breakpoints for B bronchiseptica against ampicillin, florfenicol, and tulathromycin. Bordetella bronchiseptica isolates in this study had no in vitro activity to ampicillin (0% susceptibility; susceptible breakpoint ≤ 0.5 µg/mL) in which MIC₉₀ values were 8 to ≥ 16 µg/mL. Bordetella bronchiseptica susceptibility to florfenicol (breakpoint ≤ 2 µg/mL) was low and ranged from 3.9% to 15.2% in which MIC₉₀ values were 4 to 8 µg/mL over the 5-year study period. The percentage of B bronchiseptica susceptible to tulathromycin was 99.2% to 100% (breakpoint ≤ 16 µg/mL) and the MIC₉₀ value was 8 µg/mL. While CLSI-approved susceptible breakpoints were not available, the MIC₉₀ values were determined as ≥ 8 µg/mL for ceftiofur, 1 µg/mL for danofloxacin, 1 µg/mL for enrofloxacin, ≥ 32 µg/mL for penicillin, 1 to 2 µg/mL for tetracycline, 32 to ≥ 64 µg/mL for tilmicosin, and 8 to ≥ 16 µg/mL for TMP-SMX.

P multocida

The MIC distributions, MIC₅₀ values, and MIC₉₀ values for 10 antimicrobials tested against P multocida (n = 874) are reported in Table 4. The CLSI has established clinical breakpoints for P multocida against ampicillin, ceftiofur, enrofloxacin, florfenicol, penicillin, tetracycline, tilmicosin, and tulathromycin. Pasteurella multocida susceptibility to ampicillin was very high (95.5%-100%; susceptible breakpoint ≤ 0.5 µg/mL) from 2016 to 2020, while the percentage of susceptibility to ceftiofur was 100% (breakpoint ≤ 2 µg/mL), with MIC₉₀ values at ≤ 0.03 µg/mL. Pasteurella multocida was 100% susceptible to enrofloxacin in 2016 and 2019 to 2020 (breakpoint ≤ 0.25 µg/mL) with MIC₉₀ values at 0.03 µg/mL, and P multocida isolates were highly susceptible to florfenicol (> 98%; breakpoint ≤ 2 µg/mL), penicillin (97.7%-100%; breakpoint ≤ 0.25/per mL), tilmicosin (97.6%-100%; breakpoint ≤ 16 µg/mL), and tulathromycin (99.5%-100%; breakpoint ≤ 16 µg/mL) in which the tulathromycin MIC₉₀ value ranged from 2 to 4 µg/mL. Clinical and Laboratory Standards Institute-approved susceptible clinical breakpoints have not been established for danofloxacin or TMP-SMX, but MIC₉₀ values were determined as 0.03 µg/mL and 0.12 µg/mL, respectively.

S suis

The MIC distributions, MIC₅₀ values, and MIC₉₀ values for 10 antimicrobials tested against S suis (n = 1223) are reported in Table 5. The CLSI has established clinical breakpoints for S suis against ampicillin, ceftiofur, enrofloxacin, flor-fenicol, penicillin, and tetracycline. Streptococcus suis susceptibility to ampicillin was very high (susceptible breakpoint ≤ 0.5 µg/mL) and ranged from 97.8% to 99.2%, while the percentage of susceptibility to ceftiofur was also high (91.2%-97.7%; breakpoint ≤ 2 µg/mL) over the 5-year study period in which MIC₉₀ values ranged from 1 to 2 µg/mL. The percentage of S suis susceptible to enrofloxacin (breakpoint ≤ 0.5 µg/mL) ranged from 87.3% to 94.1% in which MIC₉₀ values were 0.5 to 1 µg/mL. The percentage of S suis susceptibility to florfenicol was very high (breakpoint ≤ 2 µg/mL) and increased from 97.7% in 2016 to 100% in 2020, in which MIC₉₀ values were 2 µg/mL. The percentage of S suis susceptibility to penicillin (breakpoint ≤ 0.25 µg/mL) decreased slightly from 81.8% in 2016 to 78.6% in 2020 in which MIC₉₀ values ranged from 1 to 2 µg/mL. Streptococcus suis susceptibility to tetracycline was very low and ranged from 0.8% in 2016 to 2.1% in 2020. Susceptible breakpoints were not available for danofloxacin, tilmicosin, TMP-SMX, or tulathromycin, but MIC₉₀ values were determined as 1 µg/mL, ≥ 64 µg/mL, 0.12 to 0.25 µg/mL, and ≥ 128 µg/mL, respectively.

Discussion

The prevalence of A pleuropneumoniae, B bronchiseptica, P multocida, and S suis pathogens associated with SRD emphasizes the importance of maintaining high levels of susceptibility to antimicrobials that are available to veterinarians for treatment of these pathogens.⁹ Surveillance and monitoring studies for antimicrobial resistance in pathogenic bacteria of animal origin are necessary to understand any rates of change in the susceptibility of bacteria to antimicrobials, thereby serving as one component among many to help guide practitioners to select the most appropriate antimicrobial for treatment of disease.¹⁰

Antimicrobial resistance surveillance programs support antibiotic stewardship principles which require all antibiotic prescribers (for animals and humans) to assure good prescribing decisions that mitigate the emergence of resistance to preserve the effectiveness of antibiotics for veterinary and human medicine. Additionally, selecting the proper course of antimicrobial treatment for an animal, whether it is over-the-counter, prescribed, or through a Veterinary Feed Directive, should correlate with the Animal Medicinal Drug Use Clarification Act.   

A limited number of surveillance studies have investigated in vitro susceptibilities of specific antimicrobials used to treat swine pathogens associated with respiratory disease on a national and international basis.^4-6,11-14 The SRD surveillance program reported herein has continuously obtained swine pathogens for over 20 years from North American veterinary diagnostic laboratories that have then been tested for antimicrobial susceptibility. The purpose for this ongoing surveillance study was to summarize the antimicrobial susceptibility profiles of 2949 isolates from 4 different pathogenic bacterial species associated with SRD collected from laboratories in the United States and Canada over a 5-year period from 2016 to 2020. To our knowledge, when coupled with our published SRD surveillance data from 2001 to 2010 and 2011 to 2015, this is the only surveillance program that has collected and published 20 years of SRD susceptibility data against a total of 11,992 isolates from the United States and Canada.^4,5

Retrospective studies have been published that investigated the antimicrobial susceptibility of A pleuropneumoniae isolates from swine. Pangallo et al¹⁵ showed high antimicrobial susceptibility for 354 isolates of A pleuropneumoniae from Italy to penicillins, fluoroquinolones, tetracyclines, and ceftiofur while low rates of susceptibility were observed for florfenicol. Holmer et al¹⁶ reported the antimicrobial susceptibilities of A pleuropneumoniae from Danish pigs in which high susceptibility (> 95%) to ceftiofur, florfenicol, tulathromycin, tilmicosin, penicillin and tetracycline was observed for 135 isolates. Susceptibility data for A pleuropneumoniae from our 2001 to 2010 SRD surveillance program reported 100% susceptibility to ceftiofur, florfenicol, and tulathromycin and susceptibility data from our 2011 to 2015 SRD surveillance program reported 100% susceptibility to ceftiofur and flor-fenicol with high levels of susceptibility (> 90% to 100%) to enrofloxacin and tulathromycin.^4,5 This current report shows 100% susceptibility to ceftiofur, florfenicol, and tulathromycin along with high levels of susceptibility (> 95%) to tilmicosin, and low levels of susceptibility (0%-10.6%) to tetracycline for 250 A pleuropneumoniae isolates from 2016 to 2020. Actinobacillus pleuropneumoniae MIC values have remained high for tetracycline since 2001 and may be due to distribution of tetracycline resistance genes associated with plasmids which have been previously reported.^17,18

For B bronchiseptica, El Garch et al⁶ reported high susceptibility to amoxicillin-clavulanate (95.8%) and tulathromycin (99.2%) and lower susceptibility to flor-fenicol (52.5%). In our previous study we reported ≥ 99% susceptibility to tulathromycin, no susceptibility (0%) to ampicillin, and low susceptibility (5.4%-23.5%) to florfenicol against 572 B bronchiseptica isolates from 2011 to 2015.⁵ This current report shows ≥ 99% susceptibility to tulathromycin, 0% susceptibility (100% resistance) to ampicillin, and low susceptibility (3.9%-15.2%) to florfenicol against 602 B bronchiseptica isolates from 2016 to 2020.

For P multocida isolated from swine, El Garch et al⁶ reported 100% susceptibility to amoxicillin-clavulanate, ceftiofur, enrofloxacin, and tulathromycin and 65.8% susceptibility to tetracycline for 152 isolates. Susceptibility data from 2001 to 2010 for our SRD surveillance program reported 100% susceptibility to ceftiofur with high rates of susceptibility (> 90%-100%) to enrofloxacin, florfenicol, tilmicosin, and tulathromycin and data from our 2011 to 2015 SRD surveillance program reported 100% susceptibility to ceftiofur, enrofloxacin, and florfenicol and high levels of susceptibility (> 90%-100%) to ampicillin, penicillin, tilmicosin, and tulathromycin, with low levels of susceptibility (22.3%-35.3%) to tetracycline for 855 P multocida isolates.^4,5 This current report shows 100% susceptibility to ceftiofur along with high levels of susceptibility (> 95%) to ampicillin, enrofloxacin, florfenicol, penicillin, tilmicosin, and tulathromycin and low levels of susceptibility (23.2%-38.2%) to tetracycline for 874 P multocida isolates from 2016 to 2020.

For S suis, El Garch et al⁶ reported high susceptibility (96%-100%) to amoxicillin-clavulanate, ceftiofur, enrofloxacin, and florfenicol and 4% susceptibility to tetracycline when tested against 151 isolates. Additionally, other studies have shown high rates of resistance among S suis isolates against tetracycline (75%-100% resistance) while the year 2 report from the US Department of Agriculture’s Animal and Plant Health Inspection Service pilot project showed that of 167 S suis isolates, 2.4% were resistant to ceftiofur and enrofloxacin, 0.6% were resistant to ampicillin, 15.6% were resistant to penicillin, and 98% were resistant to tetracycline.^19,20 Susceptibility data from our 2001 to 2010 SRD surveillance program reported high rates of susceptibility (> 90%-100%) to ceftiofur and florfenicol and susceptibility data from our 2011 to 2015 report showed high levels of susceptibility (> 90%-100%) to ampicillin, ceftiofur, and florfenicol, with low levels of susceptibility (0%-1.3%) to tetracycline against 1201 S suis isolates.^4,5 This current report shows > 90% susceptibility to ampicillin, ceftiofur, and florfenicol, low levels of susceptibility (0.8%-2.1%) to tetracycline, and moderate rates of resistance among S suis to penicillin (18.2%-21.4% resistance) for 1223 S suis isolates from 2016 to 2020. Due to the inability to genetically characterize these S suis isolates, some may belong to other bacterial species, and thus the resistance rates could be affected.

Numerous authors have highlighted the challenges of surveillance programs and the potential biases that may be encountered.^5,6,21,22 While there is no “gold standard” for evaluating the antimicrobial surveillance of animal pathogens, a report is available that offers guidance on areas in which harmonization can be achieved in veterinary antimicrobial surveillance programs with the intent of facilitating comparison of data among surveillance programs.²³ All surveillance studies still have certain biases and limitations to consider when interpreting susceptibility data. For this current study, 2949 clinical isolates were collected from 2016 to 2020 and analysed, but this number of clinical isolates is still small when considering the number of SRD cases in North America over the last 5 years. As the isolates in this current study originated from many veterinary diagnostic laboratories, the methods of sample selection, collection, and submission varied among laboratories. To help decrease regional sampling bias in this study, the number of isolates of a target species from any herd was restricted to one isolate during any quarter year period.^4,5 Biases reported in other programs, such as a passive surveillance design, no consideration in differences between livestock farm types and sizes, or prior treatment of animals with antibacterial agents, are acknowledged in this and other studies.^4-6 Furthermore, the lack of clinical breakpoints or interpretive criteria for certain antibacterial agents against pathogens to determine rates of susceptibility continue to be a limitation to veterinary surveillance. A greater collaborative effort among academic and industrial veterinary groups should be made to identify what gaps exist for available breakpoints and then establish CLSI-endorsed clinical breakpoints if a standardized approach is used.

The data presented from this current study, especially data that show a continued lack of susceptibility to certain antimicrobials such as tetracycline, should serve to underscore the importance of prudent use of these drugs when treating SRD. Although tetracycline has traditionally served as the class representative agent for in vitro susceptibility testing for veterinary tetracyclines, extrapolation of tetracycline susceptibility results may not necessarily be predictive of activity or clinical outcome for other tetracycline agents, such as oxytetracycline or chlortetracycline, due to differences in blood and lung-tissue concentrations and differences in bioavailability. Even though there are CLSI-established clinical breakpoints for tetracycline that were used in evaluating data in this study, these breakpoint values were derived partly from oxytetracycline pharmacokinetic data.⁸

Management practices used in modern pig farming such as manure management, age-segregation of pigs, and nutritional and metabolic awareness have profound influences on microbial interactions which may result in decreased disease among swine.²⁴ The high levels of antimicrobial susceptibility observed in this study and others may be attributed to specific health management practices within swine herds such as the all-in, all-out management practice system. Another management practice that may be contributing to overall high antimicrobial susceptibility rates is multi-site production where contained groups of pigs spend their production life in different facilities appropriately designed for each age group (site I: breeding herd; site II: nursery; site III: finishing, all of which are located at separate geographical locations to minimize disease transmission). Future studies may be able to determine if these management practices influence antibiotic resistance changes over time, and if resistance reduction can be achieved through alterations in further enhanced housing and cleaning practices.

The results of this surveillance study when using standardized susceptibility testing methods show high percentages of antimicrobial susceptibility among the major respiratory tract pathogens isolated from swine across the United States and Canada, except for tetracycline, and results from this 5-year SRD surveillance study are similar to those previously published.^4,5 This surveillance study continues to be useful in identifying the development of antimicrobial resistance among SRD target pathogens which is crucial for the prudent use of antimicrobials in veterinary medicine. Additionally, understanding the in vitro susceptibility of SRD pathogens isolated in the United States and Canada continues to be an important component of antimicrobial stewardship and One Health.

While this study shows high rates of susceptibility for antimicrobials against SRD pathogens, public perceptions and regulatory pressures continue to drive the need for newer, alternative treatment options which may include novel antibacterial classes, re-evaluation of older or discontinued antibacterial agents, posology, and alternative approaches such as bacteriophages and peptides.²⁵

Implications

Under the conditions of this study:

• Susceptibility rates of SRD pathogens were high to key antimicrobials approved for SRD treatment.
• Antimicrobial stewardship benefits from susceptibility monitoring.

Acknowledgments

The authors want to thank the veterinary diagnostic laboratories affiliated with the following universities for providing isolates for this study: Atlantic Veterinary College, Prince Edward Island, British Columbia Ministry of Agriculture, Colorado State University, Cornell University, Iowa State University, Kansas State University, Manitoba Agriculture Services, Michigan State University, North Dakota State University, Ohio Department of Agriculture, Oklahoma State University, Pennsylvania State University, South Dakota State University, Texas A&M-College Station, University of California (Davis and Tulare), University of Guelph, University of Illinois, University of Minnesota, University of Montreal, University of Nebraska, University of Saskatchewan, University of Wisconsin (Barron and Madison), University of Wyoming, and Washington State University.

Conflict of interest

Authors Sweeney, Gunnett, Kumar, Lunt, and Galina Pantoja were employed by Zoetis and authors Bade and Machin were employed by Microbial Research, Inc at the time this study was planned and performed.

Disclaimer

Scientific manuscripts published in the Journal of Swine Health and Production are peer reviewed. However, information on medications, feed, and management techniques may be specific to the research or commercial situation presented in the manuscript. It is the responsibility of the reader to use information responsibly and in accordance with the rules and regulations governing research or the practice of veterinary medicine in their country or region.

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