The therapy of subclinical mastitis may be questionable regarding the economic benefit.148 In consideration of the reduced milk quality and milk production, but also the potential contagiousness, infections should not remain untreated.29,108 However, antimicrobial therapy should be considered as only one part of a successful treatment regime.167 The frequent removal of the altered secretion by milking is the most important accompanying measure. The application of oxytocin supports this intervention efficiently.167 The healing of the udder tissue can be promoted by anti-inflammatory symptomatical treatment.167
Dry-off treatment in dairy cows is one of the rare cases in veterinary medicine for which prophylactic treatment is indicated.167 On one hand, during this period, latent infections are likely to become acute. On the other hand, this period allows a long-lasting presence of the antimicrobial agents in the udder tissue. The use of bactericidal antimicrobial agents is recommended to diminish the development of bacterial resistance.167
3.1. Antimicrobial intervention
Antimicrobial agents and combinations belonging to the groups of the β-lactams (penicillins, cephalosporins), lincosamides, macrolides, aminoglycosides, trimethoprim/
sulfonamides and fluoroquinolones are approved for mastitis therapy, either as local or parenteral application.167
The susceptibility of the causative agent and the pharmacokinetic properties of the drug are two factors of great importance for the success of the antimicrobial therapy. The intramammary application should be preferred, if the general condition of the cow is not impaired. The permeation of parenterally applied drugs into the udder tissue is influenced by the status of the blood-udder-barrier. The permeability of this barrier increases during severe acute inflammation. Connective tissue in the chronically inflamed udder reduces the permeability.167
3.2. MLSB antibiotics
The group of MLSB antibiotics comprises three classes of drugs, the macrolides, the lincosamides and the B compounds of streptogramins (Table 2). They act bacteriostatically by inhibition of the bacterial protein biosynthesis. These drugs cause a premature dissociation of the polypeptide chain from the ribosome during the elongation step (Figure 1).101,154
Table 2. Members of MLS antibiotics
The structurally unrelated substances have been grouped, because they bind to overlapping sites within domain V of the 23S rRNA in close proximity to the peptidyl transferase centre of the large ribosomal subunit.140,168,173 Therefore, target site modification leads to cross-resistance between all classes of MLSB antibiotics. The recently approved ketolides are derivatives of the 14-membered macrolides, which have been developed to circumvent this most important resistance trait. In these drugs, the cladinose of erythromycin (Figure 2) is replaced by a keto group. Due to an additional side chain they have a strong binding affinity to a second ribosomal hairpin within domain II of the ribosome to which the binding of macrolides is very weak.50 Since this domain is not affected by the target site modification in domain V, ketolide activity was hoped to be not impaired. Unfortunately, in staphylococci their efficacy is also heavily reduced by this target site modification.150 Besides target site modification, there exists a number of other resistance mechanisms that affect only
one or two classes or even single substances. Details of bacterial resistance to MLSB
antibiotics are presented in the subchapters 5.1–5.3 of the introduction.
Among the MLSB antibiotics, only the macrolides and lincosamides play a role in veterinary medicine. Streptogramin antibiotics are currently not licensed for any veterinary application in Germany. Streptogramin antibiotics consist of two components, the A and the B compound. Both components act synergistically on different sites in the 50S subunit of the bacterial ribosome. Only the target site of streptogramin B antibiotics overlaps with that of the macrolides and lincosamides. The target site of the A compound differs and is not affected by MLSB resistance mechanisms. Streptogramin A and streptogramin B antibiotics alone act bacteriostatically; in combination, however, they exhibit bactericidal activity. Streptogramins are active against a similar spectrum of bacteria as the macrolides.
The following sections will concentrate on those classes of the MLSB antibiotics which are of interest in veterinary medicine – the macrolides and the lincosamides. Nevertheless, if B streptogramins are targeted by the same resistance trait, they will also be mentioned.
5‘ 3‘
E site P site A site
aa aa
aa aa
aa aa aa
Figure 1. Simplified scheme showing the mode of action of macrolides. The drug, represented by the hexagon, binds near the peptidyl transferase centre, blocks the growing peptidyl chain and causes its premature dissociation from the ribosome.
Macrolides. Macrolides are lactones with ring sizes of 12–16 atoms, substituted by at least two neutral or amino sugars. Members of the 14- to 16-membered macrolides are of pharmaceutical use in veterinary and human medicine (Table 2). The 14-membered prototype of this class, erythromycin (Figure 2), was discovered and isolated from Streptomyces
erythreus in 1952. Oleandomycin, clarithromycin and roxithromycin are derivatives of erythromycin with improved pharmacokinetic characteristics. Further improvement of pharmocological qualities led to the 15-membered macrolide azithromycin. Tylosin, spiramycin, tilmicosin and josamycin belong to the 16-membered macrolides. Tulathromycin, recently approved for veterinary medicine only, is a 1:9 composition of 13- and 15-membered molecules.
The macrolides do not inhibit the peptidyl transferase activity directly, but block the exit of the tunnel by which the nascent polypeptide chain passages (Figure 1).140,173 Small oligopeptides of variable length can be produced by the ribosome, depending on the substitutions at the lactone ring of the macrolide and on the sequence of the growing peptidyl chain. The 14-membered erythromycin allows the synthesis of longer peptides than the 16-membered macrolides spiramycin and tylosin do.140,173 In addition, some macrolides, including erythromycin, also inhibit the assembly of the 50S subunit of the ribosome.20,154,168,173
Figure 2. Erythromycin: a 14-membered lactone ring with a 3-cladinose and a 5-desosamine sugar.
In general, macrolides are active against Gram-positive cocci – especially staphylococci, streptococci and enterococci –, Gram-positive bacilli and Gram-negative cocci.83 Especially the newer derivatives display a broader spectrum of activity.83,134 Enterobacteriaceae are intrinsically resistant against macrolides, because the drug is not able
to penetrate their outer membrane.83 Efflux systems also seem to play a role, as it had been shown for the AcrAB-TolC system in Escherichia coli.21
Macrolides are weakly basic and lipophilic molecules. Due to this characteristic they accumulate in the udder tissue and are appropriate therapeutics for mastitis. Macrolides of use in mastitis therapy are listed in Table 3.
Table 3. MLSB antibiotics of use in mastitis therapy
Antimicrobial agent Examples
Macrolides erythromycin
tylosin spiramycin
Erythrocin vet®, Erytrotil® Tylan
Suanovila
Lincosamides lincomycin
pirlimycin Albioticb
Pirsue
a The approval for Suanovil® has been terminated in 10/2005 in Germany.
b Albiotic is a combination of lincomycin and neomycin.
Lincosamides. Lincomycin and clindamycin are composed of the amino acid proline and an S-containing octose. In pirlimycin, the proline is replaced by a piperidin derivative (Figure 3). Lincomycin was isolated from Streptomyces lincolnensis in 1962. Clindamycin, a semi-synthetic chlorinated derivative of lincomycin, shows much better tissue penetration properties. In contrast to the macrolides, the lincosamides directly interact with the peptidyl transferase centre, thereby inhibiting the correct positioning of the tRNAs, but also blocking the exit tunnel for the nascent peptidyl chain.140 The spectrum of activity resembles that of the macrolides, with the exception of the enterococci which are intrinsically resistant.154 The activity of lincosamides is enhanced in an anaerobic milieu. Especially clindamycin is also useful against anaerobes, such as Bacteroides or Clostridium.73,84
Pirlimycin was licensed in Germany in 2001 exclusively for the treatment of bovine subclinical mastitis caused by Gram-positive cocci. The advantage of pirlimycin over the previously mentioned substances is based on its pharmacokinetic characteristics. Suitable tissue permeability of pirlimycin allows its application only once a day. Lincosamides of use in mastitis therapy are listed in Table 3.
A. B. C.
Figure 3. A. Lincomycin and B. clindamycin: composed of a proline and an S-sugar; C. pirlimycin: the proline is replaced by a piperidin.