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.