By John R. Middleton, University of Missouri
Antimicrobial resistance is a major concern to physicians, veterinarians, producers, and consumers worldwide because resistance can render some diseases such as mastitis untreatable. Antimicrobial resistance is most often determined in vitro (on the bench top) by either a disk diffusion method or a broth microdilution method. By using these methods and correlating the results with clinical outcomes, it can be determined whether a bacterium will likely be susceptible or resistant to treatment when a particular drug is applied in the host. The Clinical and Laboratory Standards Institute (CLSI) publishes standards and breakpoints for estimating whether a drug will be effective or not. However, with some diseases such as mastitis, there can be a lack of correlation between in vitro antimicrobial susceptibility testing and clinical efficacy (Call et al., 2008). This can be due to site of drug delivery and subsequent distribution in the mammary gland or because many of the drugs do not have an appropriate CLSI breakpoint for mastitis. Hence, in some cases, lack of in vitro susceptibility, i.e., resistance, may incorrectly estimate our ability to effect a cure.
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Antimicrobial resistance can occur via an assortment of mechanisms including the organism being intrinsically resistant to one or more antimicrobials, by spontaneous mutation, or by transfer of genes encoding for resistance from one bacterial host to a new bacterial host via conjugation (sexual transfer of DNA), transduction (bacteriophage transfer), or transformation (acquisition and incorporation of DNA released into the bacteria’s environment by lysis of other bacteria) (Cohn and Middleton, 2010). Antimicrobials are commonly used in livestock production for treatment of disease, prophylaxis, and to improve production. When an antimicrobial drug is used, antimicrobial resistance is promoted either because there is a competitive advantage for inherently resistant bacterial strains to proliferate in the population, or use of the antimicrobial facilitates movement of resistance genes from one bacterial host to a new bacterial host (Call et al., 2008). While antimicrobials are used for a number of reasons including lameness, calf scours, and respiratory disease, the most common reason for antimicrobial use on dairy farms is to treat mastitis (Pol and Ruegg, 2007). However, despite continued prophylactic and therapeutic use of intramammary antimicrobials for the treatment and prevention of mastitis, recent reports suggest a trend toward an overall increasing susceptibility of mastitis pathogens to antimicrobials rather than increasing resistance (Erskine et al., 2002).
Mastitis can be caused by both Gram-positive and Gram-negative bacteria. However, due to the general lack of treatment efficacy against Gram-negative bacteria and a relatively high spontaneous cure rate, most antimicrobial therapies for mastitis have been targeted against Gram-positive bacteria. The most common Gram-positive bacteria isolated from cows' mammary glands are the staphylococci followed by the streptococci. Antimicrobial resistance is most common among the staphylococcal mastitis isolates with a much lower proportion of streptococcal isolates exhibiting resistance (Call et al., 2008). A particular type of antimicrobial resistance among staphylococci, methicillin resistance, has been a recent focus of mastitis researchers. The remainder of the paper will focus on methicillin-resistant staphylococci and their implications for human and animal health on the dairy farm.
Based on a recent National Animal Health Monitoring System survey (NAHMS Dairy 2007), members of the β-lactam class of antimicrobials (e.g., penicillins and cephalosporins) are the most commonly used drugs to treat subclinical and clinical mastitis and to prevent infection during the dry period in the United States. Penicillin and other β-lactam antimicrobials act by binding to a transpeptidase involved in cell wall peptidoglycan synthesis, which disrupts the bacterial cell wall, resulting in death of the bacterium. One mechanism by which bacteria become resistant to β-lactam antimicrobials is through the production of enzymes such as β-lactamase that destroy the antimicrobial’s β-lactam ring, rendering the drug ineffective. Shortly after the introduction of penicillin in the 1940s, this type of resistance became prevalent, leading to the development of new synthetic β-lactam drugs such as methicillin that resisted β-lactamase. After only a few years of use, however, Staphylococcus aureus became resistant to methicillin (Jevons, 1961). Resistance to methicillin is not mediated through production of β-lactamase, but rather methicillin-resistant staphylococci have acquired a mobile genetic element known as staphylococcal cassette chromosome mec (SCCmec). This staphylococcal cassette chromosome carries a gene known as mecA, which encodes for an altered penicillin-binding protein (PBP2a or PBP2’). The PBP2a has a lower affinity for β-lactam antimicrobials than the normal PBP such that these antimicrobials are ineffective. Importantly, the staphylococcal cassette chromosome containing the mecA gene can spread among bacteria within staphylococcal populations. Furthermore, the staphylococcal cassette chromosome contains additional insertional DNA sequences that allow for incorporation of additional antimicrobial resistance markers. These insertional sequences explain why many methicillin-resistant staphylococci are resistant to non-β-lactam antimicrobials that act through mechanisms other than interference with bacterial cell wall synthesis (e.g., macrolides, fluoroquinolones) and thus why methicillin-resistant strains can be multi-drug resistant.
While methicillin resistance is often associated with S. aureus, so-called methicillin-resistant S. aureus (MRSA), it can be commonly found in other staphylococci most frequently classified as the coagulase-negative staphylococci (CNS), and there is speculation that MRSA may have acquired the mecA gene from CNS. The moniker "methicillin-resistant" stems from the original description of MRSA in 1961 (Jevons, 1961). However, routine diagnostic screening now employs testing for in vitro susceptibility to either oxacillin or cefoxotin, as methicillin is no longer used in clinical practice. In addition to phenotypic characterization using susceptibility testing, molecular methods, i.e., polymerase chain reaction (PCR) that detect the mecA gene, are now employed to confirm a diagnosis of mecA-mediated resistance, as bacteria expressing β-lactamase may be falsely identified as mecA positive when using phenotypic methods alone. While the PCR test for confirming a diagnosis has been widely employed, recently mecA gene variants have been identified in some staphylococcal isolates from people and dairy cattle that are not detected using the current PCR test (Garcia-Alvarez et al., 2011). Hence, these mecA variants may be misclassified as methicillin susceptible using the current PCR detection method.
Methicillin-resistant staphylococcal strains are not necessarily more virulent than their methicillin-susceptible counterparts but are more difficult to treat as they are often resistant to multiple classes of antimicrobial drugs as illustrated above. Historically, MRSA was associated with hospital-acquired (HA-MRSA) infections. More recently, however, there has been an increased incidence of non-healthcare-associated, or so-called community-acquired MRSA (CA-MRSA) infections. Additionally, starting in the early 2000s, a new type of MRSA began to emerge, the so-called livestock-associated MRSA (LA-MRSA). In both humans and animals, inapparent colonization is far more common than outright infection, and colonization is more often transient than chronic. However, colonization does increase the host’s risk to MRSA infection.
MRSA in Livestock
Methicillin-resistant staphylococci have been isolated from a number of animal hosts including cats, dogs, horses, cattle, chickens, rabbits, and pigs (Cohn and Middleton, 2010). As discussed above, colonization of healthy animals with either MRSA or other methicillin-resistant staphylococci appears to be far more common than overt infection.
Staphylococci are among the most common bacteria isolated from bovine milk and cause both clinical and subclinical mastitis. S. aureus is a major contagious mastitis pathogen that is spread from cow to cow, usually during milking. The CNS are frequently isolated from milk but are generally regarded as minor mastitis pathogens. Devriese et al. (1972) were the first to describe MRSA in animals, with the detection of MRSA in mastitic cattle in Belgium. However, while S. aureus and CNS are commonly isolated from milk, methicillin-resistant staphylococci have been infrequently isolated from cases of clinical and subclinical bovine mastitis (Holmes and Zadoks, 2011). Furthermore, the origin of MRSA intramammary infections in dairy cattle was historically difficult to define. Devriese and Hommez (1975) reported that MRSA infections in dairy cattle were most likely of human origin. At that time, however, molecular strain-typing techniques were not available. With the advent of multilocus sequence typing (MLST) and spa-typing (DNA-based strain-typing methods), the host range and transmissibility between hosts have been more easily assessed. For example, a Hungarian study recently reported that MRSA isolates from dairy cattle with subclinical mastitis and a farm worker were phenotypically and genotypically indistinguishable, suggesting cross-species transmission, but still not definitively identifying the origin of infection (Juhasz-Kaszanyitzky, 2007). In addition to MRSA being associated with mastitis, a recent report documented methicillin-resistant Staphylococcus epidermidis as being isolated from a cow with mastitis on an organic dairy farm, but it was unclear whether the isolate was of bovine or human origin (Walther, 2007). While S. aureus strains seem to be somewhat host specific, humans and animals may share similar S. epidermidis strains, further complicating who infected whom.
Reports of MRSA in the swine population first emerged in the Netherlands, but swine-associated MRSA colonization and infection have now been detected across Europe, North America, and Asia with rates of colonization among pigs as high as 49% and among personnel caring for swine as high as 45%. The most common strain associated with swine colonization and infection is ST398, a strain also found to colonize people who work with swine or veal calves. This strain has also now been isolated from other domesticated animals including dairy cattle, poultry, dogs, and horses (Holmes and Zadoks, 2011). While on swine farms, pigs appear to be the major reservoir for MRSA ST398; however, a recent study reported that rats on farms can be colonized with MRSA strain ST398, suggesting that farm rodent populations may play a role in dissemination and persistence of MRSA colonization and infection in swine operations. In addition, dust samples taken from swine operations have been found to harbor MRSA, suggesting that environmental reservoirs of colonization and infection may exist. Recent reports from Europe have now shown that ST398 can establish in dairy herds, and both MRSA and methicillin-susceptible variants of ST398 have been documented (Holmes and Zadoks, 2011). Hence, while MRSA detected in bovine milk were once thought to be the result of incidental infection with human host-adapted strains, there is emerging evidence that livestock-associated MRSA may be establishing in dairy cattle and thus be a potential reservoir for human infection.
Most recently, a new mecA variant MRSA has been identified in humans and dairy cattle in various locations throughout Europe. The strain accounts for <1% of human MRSA in the United Kingdom and Denmark (Holmes and Zadoks, 2011). Because the mecA variant is not detected by routine PCR methods, some MRSA may be falsely identified as methicillin susceptible. The clinical significance and epidemiology of this variant are still not fully understood, but its detection in both humans and cattle illustrates the potential for inter-species transmission.
Interspecies Transmission of Staphylococci and MRSA
While MRSA has been documented in people and animals, when a person or animal becomes colonized or infected with MRSA, it is often asked where the infection originated. Can animals serve as a reservoir for human infection and vice versa? In general, S. aureus strains seem to be fairly host-specific, and molecular strain-typing tools have allowed a better understanding of geographical distribution and host-specificity. The host-specificity of the CNS species is less well-defined.
By definition, a zoonotic infection is an infectious disease in animals that can be transmitted to people, and the natural reservoir for the infectious agent is an animal. The opposite of zoonosis, an anthroponotic infection, is one that is spread from humans to animals. As discussed, many domestic animal species can become colonized or infected with staphylococci and MRSA and might serve as potential sources of human infection. Prior to the discovery of livestock-associated MRSA, it was generally assumed that animal colonization and infection were the result of contact with a human host-adapted isolate. Livestock-associated MRSA (primarily ST398), first discovered in pigs, appears to be an exception. Reports from the early 2000s suggest that pigs are a potential major zoonotic reservoir for MRSA, and farm workers and their families are at increased risk of infection and colonization with this strain of MRSA. Additional concern is now being voiced as dairy herds in northern Europe have now been shown to include ST398-infected cattle, and a mecA variant strain has been identified in dairy cattle and humans. That said, ST398 does not appear to be very virulent, and colonization is most often detected due to routine screening rather than patients presenting with overt clinical disease.
In conclusion, whenever we treat an animal with an antimicrobial drug, a certain selection pressure is placed on the microbial population that will ultimately select for antimicrobial resistance. Interestingly, however, while antimicrobial resistance is generally more prevalent on conventionally managed farms that employ antimicrobial therapy than on organic farms, antimicrobial-resistant bacteria can persist on organic farms after many years of antimicrobial-free management, suggesting that other factors surrounding the ecology of those bacteria on the farm are also important in the size of the antimicrobial resistance population (Call et al., 2008). Prudent use of antimicrobial drugs, i.e., only applying antimicrobials where there are clearly demonstrated production and animal welfare benefits, will reduce the selection pressure for antimicrobial resistance. Optimizing management practices that promote animal health will be essential to reducing antimicrobial usage.
Methicillin-resistant staphylococci are an uncommon cause of bovine mastitis, but evidence suggests that MRSA are emerging as pathogens of veterinary importance. Both MRSA and methicillin-susceptible S. aureus can cause a range of symptoms ranging from asymptomatic colonization to severe infection. MRSA strains are, however, more difficult to treat. Currently, bovine mastitis caused by MRSA does not appear to differ appreciably from that caused by methicillin-susceptible strains. Coagulase-negative staphylococci can commonly carry the mecA gene and may serve as a reservoir for gene transfer to S. aureus. While it appears that human strains can colonize or infect animals, the opposite scenario may also exist, particularly if the infected or colonized animal is a pig, veal calf, or dairy cow. Pasteurization should kill S. aureus and MRSA in milk, and hence food safety should be ensured unless people are drinking raw milk. Food contamination with MRSA is reported, but in most instances, the food became contaminated post-harvest by a food handler. Infection control, particularly hand hygiene, is important in preventing animal-to-human transmission and vice versa. Identification of persistently colonized personnel or animals may be necessary to prevent or alleviate some infections.
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