Introduction
Bovine respiratory disease (BRD) is the most common and costly disease affecting beef cattle in North America (Tennant et al., Reference Tennant, Ives, Harper, Renter and Lawrence2014; Magstadt et al., Reference Magstadt, Schuler, Coetzee, Krull, O'Connor, Cooper and Engelken2018). Within feedlots, BRD is responsible for approximately 75% of all morbidity and 50% of all mortality. In stocker calves, BRD occurs at a much greater frequency than is commonly seen in feedlot cattle and is estimated to be responsible for 90% of all morbidity and mortality in these operations. While multiple factors play a role in the development of BRD, bacteria, particularly Mannheimia haemolytica, are ultimately responsible for the clinical signs observed in affected cattle. For this reason, antimicrobials are a mainstay of BRD therapy. Unfortunately, antimicrobial resistance is an emerging issue in BRD pathogens and isolation of multi-drug resistant (MDR) strains of M. haemolytica has become a more frequent occurrence. The purpose of this manuscript is to review the literature as it pertains to antimicrobial resistance in common BRD pathogens, particularly M. haemolytica, and how resistance might impact the outcome of therapy in cattle diagnosed with BRD.
Resistance defined
Veterinarians are most concerned about clinical resistance, in other words, what is the probability that a specific antimicrobial will effectively treat an animal infected by a specific pathogen causing a particular disease (Apley, Reference Apley2003). The concept of clinical resistance is based on clinically derived breakpoints developed by the Clinical and Laboratory Standards Institute Veterinary Antimicrobial Susceptibility Testing Committee (CLSI VAST) using the following criteria (Apley, Reference Apley2003):
1. Range of in vitro minimum inhibitory concentration (MICs) of an antimicrobial for a representative population of a specific bacterial pathogen.
2. Pharmacokinetic/Pharmacodynamic parameters established on the basis of the relationship between drug concentration and microbial susceptibility.
3. Results of clinical trials in the target species.
For BRD, the CLSI has approved BRD-specific breakpoints for penicillin (broth dilution only), ceftiofur, danofloxacin, enrofloxacin, florfenicol, spectinomycin sulfate, tulathromycin, gamithromycin, tildipirosin, tetracycline (broth dilution only), and tilmicosin (Table 1). With these antimicrobial agents, a susceptible result indicates that the likelihood of treatment success is significantly greater than if the result indicated resistance. It is important to remember, however, that the relationship between antimicrobial susceptibility testing and clinical outcome is not perfect and these breakpoints apply only when the antimicrobial is used according to label directions and the susceptibility testing is performed using CLSI-approved methods and interpretive criteria. It is also important to realize that antimicrobial susceptibility testing does not guarantee a specific clinical result in an individual animal. Susceptibility breakpoints attempt to take an in vitro test result and extrapolate it to an in vivo response and, often, disease outcome is influenced by factors such as host immune status, variations in individual pharmacokinetic parameters, or increased disease severity/prolonged disease duration. For antimicrobials without CLSI-approved breakpoints, the interpretations have been adapted from interpretive criteria extrapolated from plasma and interstitial fluid in other species. Examples of this approach include penicillin G (disk diffusion), tetracycline (disk diffusion), potentiated sulfonamides, aminoglycosides, and erythromycin. For these antimicrobial agents, a susceptible result is certainly better than a resistant one. However, there are no data available to correlate the results of susceptibility testing and expected outcome in cattle with BRD.
Table 1. Antimicrobial-pathogen combinations with CLSI-approved breakpoints for bovine respiratory disease
a Only applies to the procaine penicillin G formulation used at 22,000 IU kg−1 IM q 24 h.
b Approved breakpoints only valid for broth dilution.
The second type of resistance is defined based on data surveilling changes in profiles of susceptibility distributions in wild-type populations of bacteria (Lubbers and Turnidge, Reference Lubbers and Turnidge2015). Rather than providing data correlated to clinical outcome, these epidemiologic cut-offs represent deviations of the MIC from the original bacterial population and can be used to indicate the appearance of resistance genes. As a result, epidemiologic cut-offs might declare resistance at an MIC that is different (often lower) than a clinical breakpoint (Lubbers and Turnidge, Reference Lubbers and Turnidge2015). For the purposes of this discussion, we will be most concerned about clinical resistance and this definition of resistance will be used throughout the rest of this manuscript.
Antimicrobial resistance in BRD pathogens
The earliest published MIC distributions for M. haemolytica established using modern diagnostic laboratory methodology and CLSI-approved breakpoints were derived from a survey of animals that died of BRD over a several year period from 1988 to 2002 (Watts et al., Reference Watts, Yancey, Salmon and Case1994). In this study, 461 M. haemolytica isolates were submitted by numerous veterinary diagnostic laboratories across the USA and Canada (Pennsylvania, Wyoming, Iowa, Washington, California, Missouri, Nebraska, Oregon, Kansas, Arizona, Texas, South Dakota, Montana, Minnesota, Oklahoma, Colorado, Utah, Saskatchewan, Alberta, and Quebec) to an Upjohn laboratory for MIC determination. The results of this study are reported in Table 2. It is important to note that the interpretive criteria for tilmicosin has changed since this study was published and the prevalence of resistance to this drug would be dramatically decreased (>90% susceptible) using currently accepted criteria.
Table 2. Antimicrobial susceptibility of M. haemolytica isolates collected from the lungs of cattle that died of BRD (from Watts et al., Reference Watts, Yancey, Salmon and Case1994)
In another study, the susceptibility of 390 M. haemolytica isolates obtained from the lungs of beef cattle that died from BRD and submitted to the Oklahoma Animal Disease Diagnostic Laboratory between 1994 and 2002 was investigated (Welsh et al., Reference Welsh, Dye, Payton and Confer2004). This study found that the susceptibility to tetracycline and spectinomycin varied over the course of the study period but was consistently low for each drug. In contrast, the susceptibility to ceftiofur and enrofloxacin remained high and relatively stable throughout the duration of the study (Table 3).
Table 3. Susceptibility of M. haemolytica obtained from the lungs of Feedlot cattle from 1994 to 2002 (from Welsh et al., Reference Welsh, Dye, Payton and Confer2004)
A landmark study from Kansas State University evaluated the prevalence of multi-drug antimicrobial resistance and antimicrobial co-resistance patterns in M. haemolytica isolated from the lungs of cattle with BRD over a 3-year period (Lubbers and Hanzlicek, Reference Lubbers and Hanzlicek2013). This work showed that, between 2009 and 2011, the proportion of isolates resistant to five or more antimicrobials increased from 5 to 35%. In addition, isolates resistant to either oxytetracycline or tilmicosin were significantly more likely to be resistant to at least one other antimicrobial class.
Several studies have investigated the prevalence of antimicrobial resistance in feedlot cattle between feedlot arrival and exit. In one study, samples obtained from 10% of animals from 30% of feedlot pens in two feedlots in southern Alberta were submitted for isolation and susceptibility testing of M. haemolytica (Klima et al., Reference Klima, Alexander, Read, Gow, Booker, Hannon, Sheedy, McAllister and Selinger2011). Swabs were collected from cattle at the time of feedlot entry and again within 30 days of feedlot exit. Over the course of the study, 409 M. haemolytica isolates were obtained and resistance to all antimicrobials tested was low, ranging from 0.2 to 3.9% with resistance to oxytetracycline being most common (Klima et al., Reference Klima, Alexander, Read, Gow, Booker, Hannon, Sheedy, McAllister and Selinger2011). In another study that sampled nearly 5500 cattle from four feedlots in Canada, deep nasopharyngeal swabs (DNP) were collected from enrolled animals at the time of arrival and again at a time point prior to feedlot exit (Noyes et al., Reference Noyes, Benedict, Gow, Booker, Hannon, McAllister and Morley2015). In this study, susceptibility to 21 different antimicrobials was evaluated for 2989 individual M. haemolytica isolates. Overall, resistance was rare, with 87% of isolates susceptible to all antimicrobials tested (Noyes et al., Reference Noyes, Benedict, Gow, Booker, Hannon, McAllister and Morley2015).
In a study from Canada evaluating resistance patterns in M. haemolytica isolated from healthy cattle and cattle with BRD, a resistant phenotype was found in 18% of M. haemolytica isolates tested (Klima et al., Reference Klima, Alexander, Hendrick and McAllister2014a). Overall, resistance was more common in isolates collected from cattle with BRD (32%) than isolates collected from healthy cattle (2%). Resistance to tetracycline was the most common phenotype observed and, generally speaking, if an isolate was resistant to one drug, it was also resistant to at least one other antimicrobial class (Klima et al., Reference Klima, Alexander, Hendrick and McAllister2014a).
Work from our laboratory evaluating the prevalence of resistance in M. haemolytica after metaphylaxis with the long-acting macrolide tulathromycin has yielded surprising results (Snyder et al., Reference Snyder, Credille, Berghaus and Giguere2017). In this study, lightweight, unweaned calves entering a stocker facility in Northeast Georgia were given tulathromycin at the time of arrival to the facility to prevent BRD. DNP were collected from each animal at the time of arrival and again 10–14 days later. For all antimicrobials except ceftiofur, there was a significant increase in the proportion of isolates classified as intermediate or resistant at the time of second sampling compared to samples collected at arrival (Snyder et al., Reference Snyder, Credille, Berghaus and Giguere2017). Of the 123 calves with M. haemolytica cultured at the time of second sampling, one (0.8%) had only pan-susceptible isolates, 30 (24.4%) had at least one isolate classified as intermediate or resistant to two antimicrobial classes (fluoroquinolones and macrolides), and 92 (74.8%) had at least one isolate classified as intermediate or resistant to three antimicrobial classes (fluoroquinolones and macrolides in addition to either phenicols or cephalosporins) (Snyder et al., Reference Snyder, Credille, Berghaus and Giguere2017). Additional work by our group evaluating efficacy and resistance in both enrofloxacin and tulathromycin in high-beef calves produced similar results (Crosby et al., Reference Crosby, Credille, Giguere and Berghaus2018).
Similar work by researchers at Mississippi State University produced comparable results. In this study, DNP were collected from calves at day 0 and then 7, 14, and 21 days after arrival processing and mass medication with tildipirosin (Woolums et al., Reference Woolums, Karisch, Frye, Epperson, Smith, Blanton, Austin, Kaplan, Hiott, Woodley, Gupta, Jackson and McClelland2018). In these calves, nearly 100% of M. haemolytica isolates collected from calves at 7, 14, and 21 days after arrival processing and exposure to tildipirosin were classified as MDR and were resistant to all drugs tested except ceftiofur (Woolums et al., Reference Woolums, Karisch, Frye, Epperson, Smith, Blanton, Austin, Kaplan, Hiott, Woodley, Gupta, Jackson and McClelland2018).
Mechanisms of multi-drug antimicrobial resistance
It is clear that resistance in BRD pathogens, particularly M. haemolytica, is on the rise. It is also clear that MDR is becoming more commonplace as well. The question is then, how does resistance to multiple antimicrobials arise after exposure to only one drug? In M. haemolytica, the primary driver for the increase in MDR strains is the integrative conjugative element (ICE) (Clawson et al., Reference Clawson, Murray, Sweeney, Apley, Dedonder, Capik, Larson, Lubbers, White, Kalbfleisch, Schuller, Dickey, Harhay, Heaton, Chitko-McKown, Brichta-Harhay, Bono and Smith2016). ICEs are mobile genetic elements that integrate into the bacterial chromosome and, under the right conditions, transfer to neighboring bacterial cells (Wozniak and Waldor, Reference Wozniak and Waldor2010). These ICEs can carry multiple genes associated with antimicrobial resistance and, with exposure to one antimicrobial drug, the ICE and the rest of the resistance genes carried can be transferred to other bacterial cells (Snyder et al., Reference Snyder, Alvarez-Narvaez and Credille2019). It is important to note that ICEs have been documented to move between different bacterial pathogens associated with BRD and are not just limited to M. haemolytica (Klima et al., Reference Klima, Zaheer, Cook, Booker, Hendrick, Alexander and Mcallister2014b).
Antimicrobial resistance: impact on therapeutic outcome
First treatment success, defined as the proportion of animals successfully responding to antimicrobial therapy at the time of first pull, has historically been high in most populations in cattle on feed. Generally, a first treatment success risk of >80% is considered acceptable. However, a recent retrospective study evaluating risk factors for treatment failure found that over 30% of cattle failed to respond to first treatment (Avra et al., Reference Avra, Abell, Shane, Theurer, Larson and White2017). In this study, high-risk calves demonstrated a greater risk of treatment failure than low-risk calves (Avra et al., Reference Avra, Abell, Shane, Theurer, Larson and White2017). Unfortunately, little work has been done to evaluate the impact of antimicrobial resistance on clinical outcome in cattle with BRD. In the one published study that the author is aware of, 62% of cattle infected with susceptible M. haemolytica isolates (n = 688) responded to treatment with tilmicosin compared to 38% of animals (n = 6) with resistant isolates (McClary et al., Reference McClary, Loneragan, Shryock, Carter, Guthrie, Corbin and Mechor2011).
Conclusions
Despite the importance of BRD to the North American cattle industry, there are few well-designed studies that evaluate antimicrobial resistance in bacterial pathogens important to this disease syndrome. The majority of published literature includes diagnostic laboratory submissions obtained from dead cattle that have been treated multiple times with multiple different antimicrobials. Nevertheless, general trends would suggest that a decrease in the susceptibility of M. haemolytica has occurred over time. Recent work suggests that antimicrobial use practices that are common within certain cattle operations might be the primary factor driving selection of resistant clones. As a result, it is critical that clinicians working with cattle recognize the importance of antimicrobial resistance in BRD pathogens and how this might affect the treatment efficacy in animals with clinical disease.
