**4. Specific antimicrobial agents**

#### **4.1 Beta-lactam agents**

### **4.1.1 Amoxicillin**

436 A Bird's-Eye View of Veterinary Medicine

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

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

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

target value, but also the potential for selecting for resistant bacterial strains.

PD component of the PK/PD relationship.

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

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

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

Pharmacokinetic-Pharmacodynamic Considerations for Bovine Mastitis Treatment 439

We performed some studies with the objective of evaluating the PK behavior of AMX trihydrate in serum and milk after its IM administration at therapeutic dose in healthy lactating cows (Mestorino et al., 1997) (Fig. 2). AMX was absorbed relatively fast after its administration (T ½ab 0.92 ± 0.10 h). Because AMX trihydrate was used, and that this is not a water soluble salt, the formulation to be administered was a suspension. This formulation allows a fraction of the AMX dose to be rapidly liberated and absorbed whereas another fraction, less soluble, is precipitated at the site of administration, and absorbed more slowly, leading to the slow elimination profile described, which allows the estimation of dosing regimens with long intervals. It should be emphasized that the profile of long-acting medication is not due to slow elimination, but a slow absorption. The drug is eliminated at normal speed, but no molecules are available at a higher rate of absorption; therefore it is

Fig. 2. AMX concentrations in serum and milk after IM administration of 15 mg.kg-1 to four

The average time of penetration into milk (T ½P) of 1.24 ± 0.09 h, was considered fast enough for an antimastitic agent. The ratio AUCmilk: AUCserum was 0.18, which is a low level of milk availability (Table 5). Beta-lactam antibiotics, due to their acidic character are more dissociated in plasma than in milk, so milk penetration is severely restricted. Serum and milk concentrations were determined until 48 h post-administration, which, considering the mentioned MIC90 of *S. aureus*, does not represent a good therapeutic tool because Cmax in milk was only 0.44 ± 0.13 µg.mL-1. It has to be remarked that the mentioned parameter was obtained in healthy animals. In mastitic animals, on the other hand, it appears logical to expect a greater penetration of AMX in the gland, because raising the pH, would permit greater dissociation of AMX in the cistern, and a process of ion sequestration with higher drug concentrations in milk. In view of the influence of the factors mentioned above, PK studies should always be performed not only on healthy animals but on sick animals too.

The most significant factor affecting the cure rates for clinical *S. aureus* mastitis is the ability of the isolate to produce *ß*-lactamase. This has also been shown by other authors (Sol et al. 2000), and could indicate either that penicillin resistant strains are more virulent than penicillin-susceptible strains, or that the antibiotics used to treat mastitis caused by

the absorption process that commands the elimination half-life.

healthy dairy cows.


Table 4. In vitro susceptibility of strains of *S. aureus* obtained from clinical and sub-clinical bovine mastitis cases by different authors.

**Resistance** 

≥0.25

≥4

≥16

≥16

≥8

≥4

≥4 76

0.25-16 ≥4 Gianneechini (2002)

**Breakpoint References** 

0.06-4 <sup>≥</sup>0.5 Gianneechini et al., 2002

Gianneechini et al., 2002 Rubin et al., 2011 San Martín et al., 2002 Russi et al., 2008

San Martín et al., 2002

San Martín et al., 2002

Gianneechini et al., 2002; Rubin et al., 2011 San Martín et al., 2002 Russi et al., 2008

Gianneechini et al., 2002 Rubin et al., 2011 San Martín et al., 2002

Gianneechini et al., 2002 Rubin et al., 2011 Russi et al., 2008

Rubin et al., 2011

Gianneechini (2002) Rubin et al., 2011 San Martín et al., 2002

Gianneechini (2002) Rubin et al., 2011

Russi et al., 2008 Lucas, 2009

Rubin et al., 2011

Gianneechini et al., 2002;

**MIC (µg.mL-1)** 

0.12 - > 8 0.06-0.5 0.062-2 0.03-8

≤0.12->16

Amoxicillin 0.125 1 0.06-4 ≥0.5 San Martín et al., 2002 AMX/CLA 2 2 2 – 16 ≥8 Rubin et al., 2011

> ≤0.5-1 0.12-0.5 0.125-16

Cephoperaz. 0.5 2 0.25-16 ≥8 San Martín et al., 2002

≤1-4 2-16 0.25-32 0.03-2

Neomycin ≤1 ≤1 ≤1-64 ≥64 Gianneechini et al., 2002

≤0.25->64 2-32 0.5-64

≤0.5->4 0.25-16 0.125-0.5

≤1

Florfenicol 0.5 2 0.25-128 ≥32 San Martín et al., 2002

≤0.25-0.5 0.25-16 0.06-8

<0.006-8 19-76

Table 4. In vitro susceptibility of strains of *S. aureus* obtained from clinical and sub-clinical

Chloramph. 4 8 4-32 ≥32 Rubin et al., 2011

Danofloxacin 0.25 1 0.25-2 Lucas, 2009

Azythromycin 0.5 1 0.25-2 Lucas, 2009 Spiramycin 4 4 0.5-4 Lucas, 2009

Cephalotin ≤4 ≤4 4-64 ≥32 Gianneechini et al., 2002;

**Antimicrobial** 

Ampicillin 0.5

Penicillin

Oxacillin Cloxacillin

Gentamicin

Oxitetracycline

Erythromycin

Enrofloxacin

Trimethoprim/ Sulfamethox

Clindamycin ≤<sup>1</sup>

**agent MIC50 MIC90 Range** 

>8 0.5 1 4

> 8 1

≤0.5 2 1

> ≤1 2 4 0.5

>64 2 4

≤0.5 0.25 0.25

≤1 0.25

≤0.25 ≤0.25 0.5

> 0.5 19

0.5 0.06 0.25 0.06

0.125

≤0.5 0.25 0.25

≤1 2 1 0.25

> 2 2 2

≤0.5 0.25 0.125

0.25

≤0.25 ≤0.25 0.125

> 0.25 19

bovine mastitis cases by different authors.

We performed some studies with the objective of evaluating the PK behavior of AMX trihydrate in serum and milk after its IM administration at therapeutic dose in healthy lactating cows (Mestorino et al., 1997) (Fig. 2). AMX was absorbed relatively fast after its administration (T ½ab 0.92 ± 0.10 h). Because AMX trihydrate was used, and that this is not a water soluble salt, the formulation to be administered was a suspension. This formulation allows a fraction of the AMX dose to be rapidly liberated and absorbed whereas another fraction, less soluble, is precipitated at the site of administration, and absorbed more slowly, leading to the slow elimination profile described, which allows the estimation of dosing regimens with long intervals. It should be emphasized that the profile of long-acting medication is not due to slow elimination, but a slow absorption. The drug is eliminated at normal speed, but no molecules are available at a higher rate of absorption; therefore it is the absorption process that commands the elimination half-life.

Fig. 2. AMX concentrations in serum and milk after IM administration of 15 mg.kg-1 to four healthy dairy cows.

The average time of penetration into milk (T ½P) of 1.24 ± 0.09 h, was considered fast enough for an antimastitic agent. The ratio AUCmilk: AUCserum was 0.18, which is a low level of milk availability (Table 5). Beta-lactam antibiotics, due to their acidic character are more dissociated in plasma than in milk, so milk penetration is severely restricted. Serum and milk concentrations were determined until 48 h post-administration, which, considering the mentioned MIC90 of *S. aureus*, does not represent a good therapeutic tool because Cmax in milk was only 0.44 ± 0.13 µg.mL-1. It has to be remarked that the mentioned parameter was obtained in healthy animals. In mastitic animals, on the other hand, it appears logical to expect a greater penetration of AMX in the gland, because raising the pH, would permit greater dissociation of AMX in the cistern, and a process of ion sequestration with higher drug concentrations in milk. In view of the influence of the factors mentioned above, PK studies should always be performed not only on healthy animals but on sick animals too.

The most significant factor affecting the cure rates for clinical *S. aureus* mastitis is the ability of the isolate to produce *ß*-lactamase. This has also been shown by other authors (Sol et al. 2000), and could indicate either that penicillin resistant strains are more virulent than penicillin-susceptible strains, or that the antibiotics used to treat mastitis caused by

Pharmacokinetic-Pharmacodynamic Considerations for Bovine Mastitis Treatment 441

MRT, which were higher in quarters of low producing-cows (*P<0.05).* The MIC90 for AMX-CLA 4:1 was 8 *µ*g.mL-1 and the T>MIC90 was higher in quarters of low-producing cows. The

It resulted remarkable that levels of AMX were higher and remained for a longer period of time in the mammary quarters of low production cows than in the quarters of high production cows. These differences were not such in the case of CLA, as calculated parameters were similar in both groups. We determined also the T>MIC90 in the milk of each quarter (MIC90= 6.4:1.6 µg.ml-1) (See Table 6). The health status of the mammary quarters had no statistically significant effect on any of the PK parameters. The level of milk production, however, had a significant effect on the AMX T½λ (P= 0.0000), AUC0-∞ (P =

IMM treatment given to supplement systemic administration of ATMs increases drug concentration in the milk compartment, and higher concentrations throughout the mammary gland will follow (Ziv 1980a; 1980c). In theory, combination treatment thus should improve cure rates for deep infections such as *S. aureus* mastitis (Sandholm et al. 1990). On the other hand, *ß*-lactam ATMs are time-dependent drugs, and very high

Fig. 3. Mean concentrations of AMX-CLA in milk of healthy and mastitic quarters after

**T>MIC90 (%)** 52.78 ± 30.71 48.60 ± 31.65 46.30 ± 35.80 53.69 ± 26.72 Table 6. T>MIC90 (%): percentage of the period between 0 and 12 h post-administration during which the concentration was ≥ the MIC90; Amx:Cla 4:1, the MIC90 was considered as

We performed another study where the effect of the combined IMM treatment with systemic administration was evaluated (Lucas et al., 2009a). The experimental animals received 3 IMM infusions of AMX-CLA (200-50 mg) in each quarter in combination with 3.5 mg.kg-1 of 15% AMX-CLA by IM route every 12 h. Individual quarter milk samples were collected until 96 h after 3rd administration. The incidence of the health status of the quarters (SS): mastitic quarters vs. healthy ones, and the level of milk production (LP): quarters of high-producing

cows vs quarters of low-producing cows in the PKs of the drugs was evaluated (Fig. 4).

**X mastitic ± SD Xhealthy ± SD XM high prod. ± SD XM low prod. ± SD** 

LP modified the AMX-CLA PK profile and it could be a determinant of efficacy.

concentrations at the infection site do not increase efficacy (Craig 2001).

administration of 3 IMM syringes (200 mg-50 mg) every 12 h

the ratio 4:1 (MIC90= 6.4:1.6 µg.mL-1)

0.0057), CL/F (P=0.0057) and MRT (P= 0.0019).

penicillin-resistant strains are less efficient, due to PK or PD factors, as we explained above. In the study by Sol et al. (2000), clinical *S. aureus* mastitis caused by *ß*-lactamase positive or negative isolates was treated intramammarily with 5 different ATM treatments. All the isolates were found to be in vitro susceptible to the drugs used.


Table 5. PK parameters (mean ± SD, N = 4) obtained in serum and milk after IM administration of AMX at dose of 15mg.kg-1 in dairy cows

No difference in the bacteriological cure rates between the different ATM treatments in each group was found, but major differences in bacteriological cure rates between mastitis due to *ß*-lactamase positive and negative strains was significant**.** *S. aureus* is known to possess many virulence factors, like capsule and slime formation, which make it more resistant to ATMs (Sandholm et al. 1990; Taponen et al., 2003).

Information about the PKs of systemic amoxicillin-clavulanic acid (AMX-CLA) suspension in dairy cows is almost totally lacking. However AMX-CLA is widespreadly used in veterinary medicine. Injectable formulations (IM) have been used in cattle, pigs and sheep, and oral formulations for the treatment of pre-ruminant animals. Like other β-lactams, AMX alone or combined with CLA is commercially available for IMM use in bovine mastitis.

There are few antecedents of combining an IMM treatment with a systemic one, and, in general these studies compare clinical efficacy without PK determinations (Ziv 1980a; 1980c). As this is an interesting strategy to treat mastitis, we decided to compare the pharmacokinetic behaviour of AMX-CLA after their IMM infusion vs the combination therapy IMM-IM in lactating Holstein cows with subclinical mastitis caused by *S. aureus* (Lucas et al., 2009a). The experimental animals were allocated by production level (high and low production level) and the quarters were grouped by health state. In the IMM alone assay, each quarter of all cows received 3 IMM infusions of AMX-CLA (200-50 mg) with a 12 h interval. Individual quarter milk samples were collected until 96 h after 3rd administration (Fig 3). The purpose was to evaluate the effects of the health status of the quarters (SS): mastitic quarters vs. healthy ones and the level of milk production (LP): quarters of highproducing cows vs quarters of low-producing cows. LP had a significant effect on T½*β* and

penicillin-resistant strains are less efficient, due to PK or PD factors, as we explained above. In the study by Sol et al. (2000), clinical *S. aureus* mastitis caused by *ß*-lactamase positive or negative isolates was treated intramammarily with 5 different ATM treatments. All the

> **Parameter Serum Milk Kabs (h-1)** 0.75 ± 0.09 0.56 ± 0.08 **T ½ abs (h)** 0.92 ± 0.10 1.24 ± 0.09 **Cmax (µg.ml-1)** 1.29 ± 0.14 0.44 ± 0.13 **Tmax (h)** 2.00 ± 0.00 4.00 ± 0.00 **ß (h-1)** 0.025± 0.07 0.03 ± 0.001 **T½ ß (h)** 27.61± 0.09 22.59± 0.91 **AUC (µg.h/ml)** 57.72± 3.87 10.34± 1.28

**R CmaxS/CmaxM** 2.93 **R AUCM/AUCS** 0.18 **FMilk (%)** 18

No difference in the bacteriological cure rates between the different ATM treatments in each group was found, but major differences in bacteriological cure rates between mastitis due to *ß*-lactamase positive and negative strains was significant**.** *S. aureus* is known to possess many virulence factors, like capsule and slime formation, which make it more resistant to

Information about the PKs of systemic amoxicillin-clavulanic acid (AMX-CLA) suspension in dairy cows is almost totally lacking. However AMX-CLA is widespreadly used in veterinary medicine. Injectable formulations (IM) have been used in cattle, pigs and sheep, and oral formulations for the treatment of pre-ruminant animals. Like other β-lactams, AMX alone or combined with CLA is commercially available for IMM use in bovine mastitis.

There are few antecedents of combining an IMM treatment with a systemic one, and, in general these studies compare clinical efficacy without PK determinations (Ziv 1980a; 1980c). As this is an interesting strategy to treat mastitis, we decided to compare the pharmacokinetic behaviour of AMX-CLA after their IMM infusion vs the combination therapy IMM-IM in lactating Holstein cows with subclinical mastitis caused by *S. aureus* (Lucas et al., 2009a). The experimental animals were allocated by production level (high and low production level) and the quarters were grouped by health state. In the IMM alone assay, each quarter of all cows received 3 IMM infusions of AMX-CLA (200-50 mg) with a 12 h interval. Individual quarter milk samples were collected until 96 h after 3rd administration (Fig 3). The purpose was to evaluate the effects of the health status of the quarters (SS): mastitic quarters vs. healthy ones and the level of milk production (LP): quarters of highproducing cows vs quarters of low-producing cows. LP had a significant effect on T½*β* and

Table 5. PK parameters (mean ± SD, N = 4) obtained in serum and milk after IM

administration of AMX at dose of 15mg.kg-1 in dairy cows

ATMs (Sandholm et al. 1990; Taponen et al., 2003).

isolates were found to be in vitro susceptible to the drugs used.

MRT, which were higher in quarters of low producing-cows (*P<0.05).* The MIC90 for AMX-CLA 4:1 was 8 *µ*g.mL-1 and the T>MIC90 was higher in quarters of low-producing cows. The LP modified the AMX-CLA PK profile and it could be a determinant of efficacy.

It resulted remarkable that levels of AMX were higher and remained for a longer period of time in the mammary quarters of low production cows than in the quarters of high production cows. These differences were not such in the case of CLA, as calculated parameters were similar in both groups. We determined also the T>MIC90 in the milk of each quarter (MIC90= 6.4:1.6 µg.ml-1) (See Table 6). The health status of the mammary quarters had no statistically significant effect on any of the PK parameters. The level of milk production, however, had a significant effect on the AMX T½λ (P= 0.0000), AUC0-∞ (P = 0.0057), CL/F (P=0.0057) and MRT (P= 0.0019).

IMM treatment given to supplement systemic administration of ATMs increases drug concentration in the milk compartment, and higher concentrations throughout the mammary gland will follow (Ziv 1980a; 1980c). In theory, combination treatment thus should improve cure rates for deep infections such as *S. aureus* mastitis (Sandholm et al. 1990). On the other hand, *ß*-lactam ATMs are time-dependent drugs, and very high concentrations at the infection site do not increase efficacy (Craig 2001).

Fig. 3. Mean concentrations of AMX-CLA in milk of healthy and mastitic quarters after administration of 3 IMM syringes (200 mg-50 mg) every 12 h


Table 6. T>MIC90 (%): percentage of the period between 0 and 12 h post-administration during which the concentration was ≥ the MIC90; Amx:Cla 4:1, the MIC90 was considered as the ratio 4:1 (MIC90= 6.4:1.6 µg.mL-1)

We performed another study where the effect of the combined IMM treatment with systemic administration was evaluated (Lucas et al., 2009a). The experimental animals received 3 IMM infusions of AMX-CLA (200-50 mg) in each quarter in combination with 3.5 mg.kg-1 of 15% AMX-CLA by IM route every 12 h. Individual quarter milk samples were collected until 96 h after 3rd administration. The incidence of the health status of the quarters (SS): mastitic quarters vs. healthy ones, and the level of milk production (LP): quarters of high-producing cows vs quarters of low-producing cows in the PKs of the drugs was evaluated (Fig. 4).

Pharmacokinetic-Pharmacodynamic Considerations for Bovine Mastitis Treatment 443

Penethamate hydriodide (PNTM) is a diethylaminoethyl ester of penicillin which, unlike salts of penicillin, is unionised and so exists in a neutral state. It is only weakly water soluble forming a suspension in an aqueous environment. After its intramuscular administration, is rapidly absorbed from the site of injection and on entering the blood, partially dissociates by hydrolysis into penicillin G and diethylaminoethanol. At the blood pH (7.4), equilibrium is established where 90% of the active drug is present in its hydrolyzed form (penicillin G) with the remainder persisting as PNTM. As PNTM leaves the circulation due to its neutral and lipophilic properties and its high affinity to milk, this equilibrium is maintained by reassociation of penicillin G and diethylaminoethanol until excretion is complete. PNTM easily passes the milk-blood barrier due to the pH gradient between milk (pH 6.6-6.8) and plasma (pH 7.2-7.4) and its weakly basic properties (pKa = 8.4). This is further facilitated by its highly lipophylic characteristics which facilitates its passage across the lipo-proteic blood-milk barrier. PNTM starts to dissociate as it passes over the barrier and this process continues during diffusion of the drug through the udder, releasing increasing quantities of penicillin G (PENG). PENG is rapidly ionised in the udder (pKa = 2.8) so limiting its return to the circulation. It therefore becomes "trapped" in the udder in increasing concentrations.

The same pH gradient between blood and milk presides in the case of mild to moderate udder inflammation such as in sub-clinical mastitis, the pH gradient between blood and milk is the same than in healthy animals thus generating similar PK behaviors to those which take place in the healthy udder. In acute mastitis, although the pH of milk is nearer that of blood due to a breakdown of the blood-milk barrier, higher concentrations of PNTM are still found in mastitic milk than in blood due to its lipophilic properties. Not only does undissociated PNTM rapidly and easily penetrate the udder whether inflammed or not, but its liposoluble nature gives it advantage, compared with other beta-lactam antibiotics such as amoxicillin and aminoglycosides to diffuse through the parenchyma of the udder, pass into the milk and penetrate the lactogenic cells. This diffusion through the udder is supported by the mechanism of "ion trapping" mentioned above and so explains the

It must be remembered, however, that *S. aureus* survives in acidic media, including phagolysosomes. Controversial in vitro/in vivo data exist on its susceptibility to antibiotics in such environments. We performed some studies to evaluate the effect of the pH variation on the antibacterial activity of penicillin against strains of *S. aureus* isolated from mastitic quarters (Moncada Cárdenas et al., 2009). MIC of *S. aureus* field strains and *S. aureus* ATCC 25923 were tested at pH 7.4, 6.5 and 5.0, in order to simulate the conditions of acidity of subcellular structures which are commonly associated with *S. aureus* intracellular persistence. The PEN MIC90 at pH 7.4 was consistent with those reported by CLSI 2007 (0.5 μg/mL) but at pH 5.0 (phagolysosomes) the activity of PENG increased markedly and

Cloxacillin (CLX) is used in the treatment or prevention of staphylococcal bovine mastitis. Its ATM activity against *S.aureus* is higher than that of PENG. There are strains of *S. aureus* resistant to isoxazolilpenicillins (oxacillin or methicillin resistant *S. aureus* –MRSA). These

different penetration of PNTM compared with PENG (Friton et al., 2003).

almost linearly (~10 fold decrease in MIC -0.06 μg/mL) (Figure 5).

**4.1.2 Penethamate** 

**4.1.3 Cloxacillin** 

strains are a menace to public health.

We determined once again the T>MIC90 in the milk of each quarter (MIC90= 6.4:1.6 µg.ml-1) (See Table 7). It was observed that concentrations achieved in cows receiving combination therapy (IM + IMM) were higher than those in cows treated intramammarly alone, which was a logical finding.

Fig. 4. Mean concentrations of AMX-CLA in milk of healthy and mastitic quarters after administration of 3 IMM syringes (200 mg-50 mg each) in combination with three 3.5 mg.kg-1 of 15% AMX-CLA IM administrations every 12 h

Significant effects of quarter health status on the PK parameters were found. The Cmax resulted higher in milk from sick quarters (P= 0.0384). The level of production had significant effect over milk AMX Cmax and Tmax, these were higher in the mammary quarters of low production cows. The level of production also affected the PK profile of CLA, the Cmax, Tmax and AUC0-∞ were higher in animals of low production. The differences observed between CL/Fmam suggest that the ATMs removal rate was higher in the quarters of high production cows. Significant effect of the mammary quarter health status and production level on the T>MIC90 was found. It should be emphasized that the T>MIC determined after the combined treatment (IM + IMM) exceeded those calculated after IMM infusion alone.


Table 7. T>MIC90 (%): percentage of the period between 0 and 12 h post-administration during which the concentration was ≥ the MIC90; Amx:Cla 4:1, the MIC90 was considered as the ratio 4:1 (MIC90= 6.4:1.6 µg.mL-1)

Owens et al. (1988) found higher cure rates in *S. aureus* mastitis with combined treatment as compared with IMM treatment only. Recently, the therapeutical effects of parenteral, IMM and combination treatments with AMX-CLA have been compared by *Perner et al.* 2002 too. They found the combination treatment to be superior than parenteral or IMM treatment only. In this paper, the bacteriological cure rate for all causing agents and mastitis types (acute, subclinical and chronic), was 75.3%. We found also low bacteriological cure rates (62.5%) after AMX-CLA IMM infusion alone, but after the combined treatment the cure rate was complete (100%) (Lucas et al., 2009a, b).
