**3.1 Post-Antibiotic Effects (PAE)**

434 A Bird's-Eye View of Veterinary Medicine

Whether a drug exhibits concentration-dependent or time-dependent killing is largely a function of the shape of its concentration-effect curve, the steeper the curve, the less will be the impact of increasing drug concentrations on the ATM response. Conversely, the more shallows the curve, the greater the relationships between the rates of bacterial kill versus the ATM drug concentration. This relationship can be described using a sigmoidal Emax model,

also known as the Hill model, which can be described as follows (Toutain, 2002):

*h* is the Hill coefficient, which adjust the degree of sigmoidicity in the curve; and

*E(t)* is the effect observed for a given concentration at time t (*C* (*t*)) ;

*Emax* is the maximal effect attributable to the drug; *EC*50 is the plasma concentration producing 50% of *Emax* ;

*E*0 describes the rate of spontaneous cure.

Gram negative organisms (Craig, 1993).

successfully control and eliminate the remaining pathogens.

Where:

function.

initial infection.

0

*E Ct Et E*

50 max ( ) ( ) ( )

When *h*= 1, the Hill model reduces to the *Emax* model, which corresponds to a hyperbolic

While there are certain characteristics common to all ATMs within a given drug class, there can be important differences in the PK/PD ratios needed to achieve a desired effect. Within the fluoroquinolones (FQ), it has been demonstrated that the rate of kill and the duration of the in vitro PAE (Finberg et al., 2004; Firsov et al., 1998b) can be markedly different across compounds and microbial species. In some cases, the PK/PD relationship necessary to achieve a 2-log kill can also vary as a function of the microbial strain (Andes and Craig, 2002). Similarly, the AUC/MIC ratio of 100-125 frequently quoted as a target for FQ ATM activity may be an appropriate predictor of success for many Gram-negative infections, but lower AUC/MIC ratios (e.g. 35 to 40) may be appropriate for infections due to Gram positive organisms (Wright et al., 2000). With regard to the β-lactams, while there tends to be a substantial in vivo PAE for *S. aureus*, a substantially shorter PAE is associated with

Within any given bacterial population, the possibilities of bacterial subpopulations that are less susceptible to the ATM agent exist. As demonstrated by Drusano (2004), unless these less susceptible pathogens are killed, succeeding microbial generations will re-populate the infection site with pathogens whose MIC values are higher than those found within the

Accordingly, ensuring adequate exposure following an initial dose of a FQ is as important as insuring that high drug concentrations occur after repeated administration. Drug concentrations need to be adequate to either destroy the existing bacterial population at the site of the infection or to reduce its size to the point where the host defense mechanism can

For drugs exhibiting concentration-dependent killing, Cmax/MIC ratios may be particularly important when the pathogen has a high MIC value or is rapidly proliferating (Craig and Dalhoff, 1998). Rapidly proliferating bacteria have a greater likelihood of undergoing a

*EC C t*

*h h h*

(4)

High drug concentrations relative to the MIC may contribute to an increase in the duration of the in vitro and in vivo PAE. For those bacteria/drug combinations that exhibit a PAE, in vivo PAEs have been shown to be longer than in vitro PAEs for most organisms. Thus, optimizing the Cmax/MIC ratio will delay the re-growth of the pathogen, sometimes by several hours. This type of dosing schemes results in fewer organisms remaining that can evolve into a resistant subpopulation and can be managed by the host defenses.

For many compounds, the duration of the in vivo and in vitro PAE is substantially greater for Gram-positive than for Gram-negative pathogens. Because the duration of the in vitro and in vivo PAE of β-lactams tends to be negligible for Gram-negative species, it is recommended that concentrations of drug remain above the MIC of the pathogen for >80% of the dosing interval to combat this type of organisms. While a T>MIC of about 40% is sufficient for staphylococcal species.

This difference in the duration of the in vitro and in vivo PAE may also be one of the reasons why the in vivo AUC/MIC for FQ tends to be less for Gram-positive than for Gramnegative pathogens. For Gram-negative organisms, the estimated AUC/MIC ratios needed to ensure effective treatment and prevent the selection of resistant strains is estimated to be approximately 100 to 125 (Forrest et al., 1993). In contrast, the AUC/MIC ratio for Grampositive bacteria is considerably lower, approximately 30 to 50 for a number of drugpathogen combinations (Wright et al., 2000). Studies involving the third and fourth generation FQ suggest that for Gram-positive organisms AUC/MIC values are substantially lower when Cmax/MIC values are ≥10 (Nightingale et al., 2000).

Blood concentrations and MIC data alone cannot predict drug effectiveness. For example, using human and bovine estimated breakpoints for cephapirin and oxytetracycline, Constable and Morin (2002) showed that the MIC values predicted that the causative pathogens would be susceptible to both agents. However, these compounds were not effective in the treatment of acute bovine mastitis. In the same line, compounds effective in the treatment of acute bovine mastitis may be ineffective in the treatment of chronic bovine mastitis (Owens et al., 1997).

Pharmacokinetic-Pharmacodynamic Considerations for Bovine Mastitis Treatment 437

decrease with increasing age of the cow, increasing somatic cell count, increasing duration of infection, increasing bacterial colony counts in milk before treatment, and increasing number of quarters infected. *S. aureus* mastitis in hind quarters has a low cure rate compared with front quarters. ATM treatment of IMMI with penicillin-resistant *S. aureus*  strains results in a lower cure rate for treatment with either *β*-lactam or non-*β*-lactam antibiotics. The most important treatment factor affecting cure is treatment duration. Increased duration of treatment is associated with increased chance of cure. Economically, extended treatment is not always justified, even when indirect effects of treatment such as

The *β-*lactams (penicillins and cephalosporins) have become the first line of ATM agents used for treatment of bovine mastitis in Argentina. Within this class, penicillin, amoxicillin, cloxacillin and ampicillin are the most used agents. In the Nordic countries penicillin is used as the first-line antibiotic treatment of bovine mastitis, because of its potency, low resistance rate and narrow spectrum. This is an important tool to limit the development of antibiotic resistance as much as possible. In a one study performed for us 34% of *S. aureus* were classified as penicillin resistant, while 100% were classified as sensible to the combination amoxicillin/clavulanic acid (Lucas, 2009a). This prevalence of resistance to penicillin was similar than those obtained by others authors in different regions of Argentina (Gentilini et al., 2000; Russi et al., 2008). And this was the same comparing the proportion of resistance (47 %) as obtained by Gianneechini et al. (2002) in Uruguay. The comparison between these results obtained over the years demonstrated that the situation in general has not changed during the last 25 years in relation to penicillin resistance. However, it results higher than in Norway, 4.2% from clinical cases and 18% from sub-clinical cases (Hofshager et al., 1999), and Sweden, 6% (Franklin, 1998). In the Table 4 we present in vitro susceptibility results of

strains of *S. aureus* obtained by different studies performed in different countries.

antibacterial activity similar to ampicillin (AMP) (Hunter et al., 1973).

AMX or AMP alone can be expected only for *S. aureus*.

The use of β-lactam antibiotics in the treatment of mastitis remains one of the first elections. Within this group, any penicillin (PEN) such as amoxicillin (AMX) provides a number of advantages. AMX is a β-lactam antibiotic of wide spectrum with a chemical structure and

AMX is rapidly converted to AMP in the body. AMX pharmacokinetics is essentially similar to other β-lactam antibiotics. The half-life is short (about 1 hour) and the volume of distribution low. The first AMX pharmacokinetic study was conducted in ruminants after IV and IM

Since then AMX disposition was studied after its administration by different routes in production animals (Archimbault et al, 1981; Baggot, 1988, Nouws et al, 1986, Wilson et al, 1988). According to Erskine et al. (2002) about 60% of *S. aureus* isolates are susceptible to AMP. The susceptibility to AMP among mastitis causing streptococci is 100% and for *E.coli*  85%. Therefore, significant improvement by AMX/clavulanic acid treatment compared to

administration in cattle and after IM administration in goats by Ziv and Nouws (1979).

**4. Specific antimicrobial agents** 

**4.1 Beta-lactam agents** 

**4.1.1 Amoxicillin** 

prevention of contagious transmission are taken into consideration.

Drug potency is often considered in terms of MIC, which is a measure of a drug´s static effect on microbial growth. The MIC may not be the same as a compound´s minimum bactericidal concentration (MBC). Since both the MIC and MBC values are in vitro estimates, they do not reflect the killing rate of a drug, the effect of serum on ATM activity, PAE, or post-ATM sub-MIC (Craig and Dalhoff, 1998). Firsov et al. (1999) demonstrated that even with comparable AUC/MIC ratios, different FQ can have marked differences in in-vitro time-kill profiles. Such differences cannot only influence the selection of an AUC/MIC target value, but also the potential for selecting for resistant bacterial strains.

Traditional in vitro susceptibility data reflect the impact of therapeutic agents on bacteria that are in the active growth phase. These tests do not describe the differential activity across the different life phases of bacteria (Cerca et al., 2005). As an example, in the case of bacteria from biofilms, entering a non-growth phase, many compounds begin to lose their ATM effects. This very important facts cannot be predicted when MIC alone is used as the PD component of the PK/PD relationship.

There are some examples of traditional susceptibility tests failing to adequately reflect in vivo drug activity. For instance, *β*-lactams are inactivated by purulent material due to the accumulation of bacterial *β*-lactamases; gentamicin can be inactivated by reversibly binding to DNA released from lysed neutrophils; netilmicin and amikacin are inactivated by disrupted leukocytes (Labro, 2000). Owens et al. (1997) noted that the bacteriologic cure rate for newly (less than two weeks duration) acquired *S. aureus* IMMI, was 70% when treated with penicillin and novobiocin combination. However, the cure rate dropped to less than 35% for chronic infections (those lasting longer than four weeks). Accordingly, they observed that while the successful treatment of acute infections could be predicted on the basis of in vitro susceptibility test results, this was not the case for chronic *S. aureus* infections. It is not know if this outcome is due to the driving of *S. aureus* infections from extracellular to intracellular sites, to biofilm formation, or to some other bacterial pathogen-host interaction (Brouillete et al., 2004).

Staphylococcal bovine mastitis exemplifies a persistent infection that is difficult to treat and where frequent relapses commonly lead to a chronic disease (Sandholm et al., 1990). Antibiotic treatment of this disease is problematic (as mentioned in the preceding paragraphs) partly because in vitro antibacterial susceptibility is a poor predictor of efficacy in chronic cows (Owens et al., 1997). Failure to eliminate the infection from dairy herds is likely due to a combination of an adaptive response of the pathogen to survive in the mammary gland despite the presence of antibiotics (Sandholm et al., 1990), and the inability of host defenses to clear the pathogen. Infectious bacteria in chronic mastitis may survive intracellularly and remain quiescent and protected from the action of antibacterials and host defenses. Although reinfections that follow attempts to treat acute mastitis can be due to newly acquired strains, it is equally possible that the reinfection results from persistence of the original infective organism (Sandholm et al., 1990). It is possible that *S. aureus* having the small-colony variants (SCV) phenotype contribute to chronic mastitis. SCVs have been isolated from cases of bovine mastitis (Ziv & Sompolinsky, 1976; Sompolinsky et al., 1969).

Both lactational and dry cow therapy are part of *S. aureus* control programs. Reported cure rates for *S. aureus* mastitis vary considerably in a range from 4 to 92% (Owens et al., 1988, 1997). The probability of cure depends on cow, pathogen, and treatment factors. Cure rates decrease with increasing age of the cow, increasing somatic cell count, increasing duration of infection, increasing bacterial colony counts in milk before treatment, and increasing number of quarters infected. *S. aureus* mastitis in hind quarters has a low cure rate compared with front quarters. ATM treatment of IMMI with penicillin-resistant *S. aureus*  strains results in a lower cure rate for treatment with either *β*-lactam or non-*β*-lactam antibiotics. The most important treatment factor affecting cure is treatment duration. Increased duration of treatment is associated with increased chance of cure. Economically, extended treatment is not always justified, even when indirect effects of treatment such as prevention of contagious transmission are taken into consideration.

The *β-*lactams (penicillins and cephalosporins) have become the first line of ATM agents used for treatment of bovine mastitis in Argentina. Within this class, penicillin, amoxicillin, cloxacillin and ampicillin are the most used agents. In the Nordic countries penicillin is used as the first-line antibiotic treatment of bovine mastitis, because of its potency, low resistance rate and narrow spectrum. This is an important tool to limit the development of antibiotic resistance as much as possible. In a one study performed for us 34% of *S. aureus* were classified as penicillin resistant, while 100% were classified as sensible to the combination amoxicillin/clavulanic acid (Lucas, 2009a). This prevalence of resistance to penicillin was similar than those obtained by others authors in different regions of Argentina (Gentilini et al., 2000; Russi et al., 2008). And this was the same comparing the proportion of resistance (47 %) as obtained by Gianneechini et al. (2002) in Uruguay. The comparison between these results obtained over the years demonstrated that the situation in general has not changed during the last 25 years in relation to penicillin resistance. However, it results higher than in Norway, 4.2% from clinical cases and 18% from sub-clinical cases (Hofshager et al., 1999), and Sweden, 6% (Franklin, 1998). In the Table 4 we present in vitro susceptibility results of strains of *S. aureus* obtained by different studies performed in different countries.
