Antibiotic dry buffalo therapy: effect of intramammary administration of benzathine cloxacillin against Staphylococcus aureus mastitis in dairy water buffalo.

BACKGROUND: Mastitis is one of the most costly diseases in Mediterranean buffalo (MB). At present, just a few specific antibiotics registered for this dairy specie have been synthetized. Efficacy of an antibiotic dry buffalo therapy (aDBT) against Staphylococcus aureus (S. aureus) mastitis, based on intra-quarter administration of 600 mg of benzathine cloxacillin, have been evaluated for the first time. Eighty MB's quarters received a drying-off therapy (aDBT-group) and 80 were left untreated (no-aDBT-group). They were sampled at drying-off (pre-treatment) and at the resumption of milking [< 10 days in milk (DIM)]. Fresh calver mastitis rate, dry period new mastitis rate, dry period cure rate, and persistent mastitis rate were calculated for clinical monitoring. Overall proportion of positive quarters/animals, quarters affected by mastitis or intramammary infections (IMI), effects on somatic cell count (SCC) and milk yield were also assessed. RESULTS: An inter-group difference (aDBT vs. no-aDBT) was recorded for all the indexes considered. An intra-group (drying-off vs. < 10 DIM) difference was detected in aDBT-group regarding the proportion of positive-cultured quarters and animals. Concerning the latter, an inter-groups difference was also recorded at second sampling. No clinical mastitis due to the S. aureus was observed. Regarding the subclinical ones, a higher intra-group difference was observed in aDBT than no-aDBT group, while an inter-group difference was recorded at second sampling. No protective effect was observed against IMI. SCC showed an inter-group difference at second sampling, while none difference was instead detected for milk yield. CONCLUSIONS: The effects against S. aureus mastitis of benzathine cloxacillin administration at drying-off were assessed for the first time in MB. Its use shows encouraging results in reducing the proportion of mastitis and positive animals at the resumption of the lactation.

Randomized noninferiority study evaluating the efficacy of 2 commercial dry cow mastitis formulations.

The study objective was to compare the efficacy of 2 commercial dry cow mastitis formulations containing cloxacillin benzathine or ceftiofur hydrochloride. Quarter-level outcomes included prevalence of intramammary infection (IMI) postcalving, risk for cure of preexisting infections, risk for acquiring a new IMI during the dry period, and risk for clinical mastitis between dry off and 100 d in milk (DIM). Cow-level outcomes included the risk for clinical mastitis and the risk for removal from the herd between dry off and 100 DIM, as well as Dairy Herd Improvement Association (DHIA) test-day milk component and production measures between calving and 100 DIM. A total of 799 cows from 4 Wisconsin dairy herds were enrolled at dry off and randomized to 1 of the 2 commercial dry cow therapy (DCT) treatments: cloxacillin benzathine (DC; n=401) or ceftiofur hydrochloride (SM; n=398). Aseptic quarter milk samples were collected for routine bacteriological culture before DCT at dry off and again at 0 to 10 DIM. Data describing clinical mastitis cases and DHIA test-day results were retrieved from on-farm electronic records. The overall crude quarter-level prevalence of IMI at dry off was 34.7% and was not different between treatment groups. Ninety-six percent of infections at dry off were of gram-positive organisms, with coagulase-negative Staphylococcus and Aerococcus spp. isolated most frequently. Mixed logistic regression analysis showed no difference between treatments as to the risk for presence of IMI at 0 to 10 DIM (DC=22.4%, SM=19.9%) or on the risk for acquiring a new IMI between dry off and 0 to 10 DIM (DC=16.6%, SM=14.1%). Noninferiority analysis and mixed logistic regression analysis both showed no treatment difference in risk for a cure between dry off and 0 to 10 DIM (DC=84.8%, SM=85.7%). Cox proportional hazards regression showed no difference between treatments in quarter-level risk for clinical mastitis (DC=1.99%, SM=2.96%), cow-level risk for clinical mastitis (DC=17.0%, SM=15.3%), or on risk for removal from the herd (DC=10.7%, SM=10.3%) between dry off and 100 DIM. Finally, multivariable linear regression with repeated measures showed no overall no difference between treatments in DHIA test-day somatic cell count linear score (DC=2.19, SM=2.22), butterfat test (DC=3.84%, SM=3.86%), protein test (DC=3.02%, SM=3.02%), or 305-d mature-equivalent milk production (DC=11,817 kg, SM=11,932 kg) between calving and 100 DIM. In conclusion, DC was noninferior to SM in effecting a cure, and there was no difference in efficacy between these 2 DCT formulations as related to all other udder health or cow performance measures evaluated between dry off and 100 DIM.

Tissue distribution of cloxacillin after intramammary administration in the isolated perfused bovine udder.

BACKGROUND: Various intramammary suspensions containing cloxacillin benzathine are registered for use in cattle as antibiotics for intramammary use at drying off. To ensure antibacterial efficacy, the glandular tissue concentration of an antimicrobial agent must be sufficient. Since the possibilities to measure concentrations in the different areas of the glandular tissue in vivo are very limited, it was the aim of the present study to examine the distribution of cloxacillin in vitro using the isolated perfused bovine udder. METHODS: Mammary glands taken at slaughter from healthy lactating cows were perfused in vitro with warmed and gassed Tyrode solution. 600 mg cloxacillin benzathine were administered as Orbenin Extra Dry Cow by the intramammary route to six front and rear quarters each. Samples of glandular tissue--at different distances from and vertical to the teat right up to the udder base--were gathered from the treated quarters after 6 h. Perfusate was also sampled before and hourly after treatment for 6 h. The cloxacillin content of the tissue samples and perfusate samples was analysed by high performance liquid chromatography. RESULTS: The concentration of cloxacillin in the glandular tissue of front quarters measured 6 h after administration tended to decrease with increasing vertical distance from the teat. The decrease pattern of the concentration was not quite clear in rear quarters. A considerable variation in the tissue concentrations of cloxacillin was obvious, which reflects in vivo conditions. The concentrations measured in the perfusate samples were below the limit of quantification at all time points, indicating limited absorption of the antibiotic from the glandular tissue. CONCLUSION: After intramammary administration of the dry off product containing cloxacillin benzathine concentrations of more than 0.5 µg/g (MIC) were reached in all regions of the front and rear quarters.

Cloxacillin benzathine-loaded polymeric nanocapsules: Physicochemical characterization, cell uptake, and intramammary antimicrobial effect.

The present work shows the development and evaluation of the veterinary antibiotic cloxacillin benzathine (CLOXB) loaded into poly-ε-caprolactone (PCL) nanocapsules (NC), as a potential new treatment strategy to manage bovine intramammary infections, such as mastitis. Staphylococcus aureus-induced mastitis is often a recurrent disease due to the persistence of bacteria within infected cells. CLOXB-PCL NC were prepared by interfacial deposition of preformed biodegradable polymer followed by solvent displacement method. The mean diameter of NC varied from 241 to 428 nm and from 326 to 375 nm, when determined by dynamic light scattering and by atomic force microscopy, respectively. The zeta potential of NC was negative and varied from -28 to -51 mV. In vitro release studies from the NC were performed in two media under sink conditions: PBS with 1% polyethylene glycol or milk. A reversed-phase HPLC method was developed to determine the NC entrapment efficiency and kinetics of CLOXB release from the NC. Free CLOXB dissolution occurred very fast in both media, while drug release from the NC was slower and incomplete (below 50%) after 9 h. CLOXB release kinetics from polymeric NC was fitted with the Korsmeyer-Peppas model indicating that CLOXB release is governed by diffusion following Fick's law. The fluorescence confocal microscopy images of macrophage-like J774A.1 cells reveal NC uptake and internalization in vitro. In addition, antimicrobial effect of the intramammary administration of CLOXB-PCL NC in cows with mastitis resulted in no clinical signs of toxicity and allowed complete pathogen elimination after treatment. The in vivo results obtained in this work suggest that CLOXB-PCL NC could be a promising formulation for the treatment of intramammary infections in cattle, considering their physicochemical properties, release profiles and effects on bovine mastitis control.

Influence of the genotype of Staphylococcus aureus, determined by pulsed-field gel electrophoresis, on dry-period elimination of subclinical mastitis in Canadian dairy herds.

By combining information from 2 databases, we investigated the possibility of an association between the genotype of Staphylococcus aureus causing bovine intramammary infection and dry-period cure of subclinical infection. The 1st database contained bacteriologic and cow data from a field study evaluating the efficacy in such infections of a new intramammary dry-cow therapy (DCT) containing tilmicosin phosphate, in comparison with a commercially available DCT containing benzathine cloxacillin. Isolates of S. aureus from that study were frozen and later independently analyzed by pulsed-field gel electrophoresis (PFGE) and macrorestriction DNA fingerprinting. The molecular information, summarized and published elsewhere, constituted the 2nd database. Data from 121 subclinically infected quarters of 92 cows from 40 herds were studied by univariate and multivariable regression analysis. Infection by an isolate of PFGE lineage group D was more likely than infection by an isolate of group A or F to be cured (P < 0.05). Cows infected by lineage group D had a higher linear somatic cell count score (LS) from the last Dairy Herd Improvement test before the dry period than did cows infected by the other lineage groups (P = 0.04). Although the probability of cure was significantly lower for cows with an LS at or above the mean of 5.7 for the study population (P = 0.05), when such a cow was infected with lineage group D, cure was significantly more likely (P < 0.001) than when it was infected by another lineage group. Significantly more (P = 0.02) of the infections treated with tilmicosin (74%) than of those treated with benzathine cloxacillin (53%) were cured, and significantly more (P = 0.05) of the infections by group D (81%) than of those by group A (57%) or group F (54%) were cured. However, there was no difference in cure rate for any PFGE genotype when tilmicosin phosphate was administered; when benzathine cloxacillin was administered, 87% of lineage group D isolates were eliminated, as compared with 46% of group A and 33% of group F isolates (P < 0.05). This research demonstrates that certain genotypes of S. aureus may naturally elicit a greater inflammatory response, yet be more susceptible to elimination by antibiotics in the dry period, than other genotypes.

Floxacillin-induced cholestatic hepatitis with evidence of lymphocyte sensitization.

A severe and prolonged form of biopsy-proved cholestatic hepatitis occurred in a 45-year-old man who had received floxacillin for two weeks preceding the episode of drug-related cholestatic injury. Immunologic tests revealed evidence of in vitro sensitization to the drug as well as to the serum of a normal subject collected after ingestion of floxacillin. Floxacillin should be added to the list of drugs causing cholestatic hepatitis, most likely by an immunologic mechanism.

Factors influencing the biological inactivation of high protein bound beta-lactam antibiotics in vitro.

Unpredictable inactivation of antimicrobial agents may cause erratic results in pharmacokinetic studies. In this study we followed the inactivation of the high protein bound beta-lactams flucloxacillin, dicloxacillin and ceftriaxone in vitro. The antibiotics were added to pools of human and rabbit sera, ultrafiltrates of these pools, rabbit interstitial fluid, phosphate buffered saline (PBS), rabbit albumin in PBS and sodium dodecyl sulphate (SDS) treated preparations of human sera. Ceftriaxone was relatively stable but different serum pools varied significantly in their flucloxacillin and dicloxacillin inactivating capacity. The dominating inactivation took place within five minutes after the addition of antibiotics to serum. The inactivating factor was heat stable at 56 degrees C, 0.5 h, of relatively high molecular weight, and not related to albumin. The inactivating capacity could be diminished by SDS-treatment of serum suggesting a lipoprotein nature.

Flucloxacillin in bone.

Ten patients undergoing total hip replacement for osteoarthritis were each given intramuscular flucloxacillin about two hours preoperatively; bone and serum were sampled simultaneously at operation. Trabecular and compact bone were separated, partly dried, reduced to powders, and then extracted with buffer. The concentration of flucloxacillin in bone washings and serum was determined by well-diffusion assay. The mean concentration of flucloxacillin in serum was 8.9 mg/l, in trabecular bone washings, 1.3 mg/l, and in compact bone washings 0.9 mg/l. The amount of blood contaminating the bone washings was measured, and was calculated to account for at most 26% of the flucloxacillin present. The significance of these findings is discussed in relation to the prophylactic use of flucloxacillin in hip replacement surgery.

Antitoxin production in antibiotic-associated colitis?

The production of antitoxin after Clostridium difficile-induced diarrhoea has not been reported previously. The stool of a patient with prolonged antibiotic-associated diarrhoea contained C. difficile toxin, and the serum neutralised the cytopathic effect of C. difficile toxin in tissue culture.

Further evolution of a strain of Staphylococcus aureus in vivo: evidence for significant inactivation of flucloxacillin by penicillinase.

A strain of Staphylococcus aureus (no. FAR4) has been isolated at intervals, for 32 months, from the sputum of a patient with cystic fibrosis of the lung. Changes in the properties of isolates of this strain over the first 18 months have been reported previously (Lacey et al., 1973 and 1974). During the last 14 months (May 1973 to July 1974), further evolution has occurred to produce a total of 31 distinct phenotypes. Recent changes are as follows. 1. The ability of isolates to produce penicillinase in vitro was closely correlated with flucloxacillin therapy. Inactivation of flucloxacillin by penicillinase was demonstrated by diffusion testing (but not MIC determination) in vitro and may have occurred to a significant extent in vivo. 2. Lincomycin-resistant mutants slowly disappeared from the sputum after the termination of clindamycin therapy. 3. All of the recent isolates were resistant to erythromycin, possibly because of the linkage of the genes coding for erythromycin resistance with those coding for the production of delta-haemolysin; delta-haemolysin may be an important "virulence factor".

A clinical trial of teicoplanin compared with a combination of flucloxacillin and tobramycin as antibiotic prophylaxis for cardiac surgery: the use of a scoring method to assess the incidence of wound infection.

A prospective randomized clinical trial is in progress to compare the efficacy of teicoplanin with flucloxacillin and tobramycin in the prevention of endocarditis and wound infection following cardiac surgery. To date, 198 patients have completed the trial, of whom 95 have received teicoplanin and 103 flucloxacillin and tobramycin. One patient developed prosthetic valve endocarditis 3 months after surgery covered by flucloxacillin and tobramycin. There was no significant difference in the incidence of sternal wound infection (P = 0.15). Severe sternal sepsis occurred in four patients in the teicoplanin group and two in the flucloxacillin/tobramycin group. There were more postoperative urinary tract infections among those given teicoplanin (15 of 95 patients compared to six of 103 patients P less than 0.05). The trial continues.

Flucloxacillin in the treatment of infectious conditions in children.

A multi-centre study was carried out in 107 children with skin and soft-tissue infections (46) or upper respiratory tract infections (61) to assess the effectivness of flucloxacillin (125 mg. q.d.s. for 5 days). Swabs were taken from the lesion sites before and after treatment for bacteriological assessment and sensitivity of the isolated organisms. The clinical success rate achieved was 93% in skin and soft-tissue infections and 94% in upper respiratory tract infections and these results correlate closely with the bacteriological findings. The main causative organism in the skin and soft-tissue infections was Staph. aureus which was resistant to benzyl penicillin and to ampicillin in all but 2 cases. All strains proved sensitive to flucloxacillin. Minimal side-effects were reported and the syrup presentation was well accepted.

Bactericidal activity and pharmacology of flucloxacillin.

Flucloxacillin, a recent addition to the group of isoxazolyl penicillins, was studied in vitro and in normal volunteers. The bactericidal activity of the drug against most strains of gram-positive bacteria including penicillin-resistant Staphylococcus aureus was similar to that of oxacillin and approximately fourfold greater than that of cloxacillin. Each of the three penicillins was administered orally to a group of ten volunteers for eight days in a dose of 500 mg four times a day. The mean concentrations of flucloxacillin in the serum were two- to sixfold higher than those of the other two agents on the first, fourth and eighth days of therapy. The percentage of flucloxacillin bound by serum protein was 94.6 per cent; for cloxacillin and oxacillin the values were 93.5 and 91.5 per cent, respectively. Using these data, the concentrations of free flucloxacillin in serum were found to be twice as high as those of cloxacillin and oxacillin. These findings suggest that, when administered orally, this new agent may offer some therapeutic advantage over oxacillin and cloxacillin.

Conversion of cloxacillin into a progressive inhibitor of beta-lactamases by sulfonation and its activity against various types of these enzymes.

On the assumption of the theory that the sulfone of a penam derivative should effectively act as a suicide substrate against beta-lactamases to which the parent compound is a poor substrate, the action of cloxacillin sulfone on four different types of beta-lactamases was studied. As we expected, cloxacillin sulfone showed strong mechanism-based irreversible inactivation against type Ib penicillinase and Proteus vulgaris cephalosporinase whereas it showed no progressive inactivation against cloxacillin-hydrolyzing type II penicillinase. However, an unexpected result was that cloxacillin sulfone could not inactivate Citrobacter freundii cephalosporinase which itself could hardly hydrolyze the parent cloxacillin. The number of hydrolytic events which occurred before inactivation of type Ib penicillinase, and P. vulgaris cephalosporinase, by the inactivator was 190 and 13, respectively. These values indicate that cloxacillin sulfone is far more effective as a suicide substrate against the two types of beta-lactamases than penam sulfones so far reported. The inactivation proceeded via the formation of an irreversible enzyme-inhibitor complex which could be detected by isoelectric focusing.

Delayed inactivation of Citrobacter freundii cephalosporinase by 6 beta-[3-(2-chlorophenyl)-5-methyl-4-isoxazolyl]penicillin sulfone.

When Citrobacter freundii cephalosporinase was incubated with 6 beta-[3-(2-chlorophenyl)-5-methyl-4-isoxazolyl]penicillin sulfone (cloxacillin sulfone) in phosphate buffer, the enzyme was suddenly inactivated just after the completion of enzymatic degradation of the cloxacillin sulfone. Such delayed inactivation was due to a secondary inhibitor formed from cloxacillin sulfone during the incubation period. The inactivation was delayed due to the protection of the enzyme by cloxacillin sulfone from the attack of the secondary inhibitor. Phosphate anions were essential for the formation of the secondary inhibitor. However, once the secondary inhibitor was formed, the inactivation occurred in the absence of phosphate anions although the degree of the inactivation depended on the length of the preincubation period with phosphate anions. The main species (more than 80%) of the inactivated enzyme was detected as a single protein band with a slightly lower pI value than that of the native enzyme on isoelectric focusing on a plate.

The penetration of antibiotics into the normal intervertebral disc.

The role of antibiotics in the treatment of disc-space infection is controversial. This study assessed the tissue penetration of flucloxacillin and cephradine into the normal intervertebral disc after intravenous administration of a bolus dose of antibiotic. Twenty-five discs were removed from 12 adolescent patients having anterior spinal surgery to correct scoliosis; antibiotic had been administered between 30 minutes and four hours before operation. Despite high blood levels, no antibiotic could be detected by bioassay or by high-pressure liquid chromatography (h.p.l.c.) in any of the specimens from the nucleus pulposus or the annulus fibrosus.

19F NMR spectroscopy study of the metabolites of flucloxacillin in rat urine.

19F NMR spectroscopy was used to monitor the metabolites of flucloxacillin in the urine of a rat dosed with its sodium salt. 19F NMR signals were detected and quantified from flucloxacillin and three metabolites. The 19F NMR method involves minimal sample preparation and is free from interference by endogenous urine components. High-field spin-echo 1H NMR spectroscopy and HPLC were used to confirm the results.

A micromethod for determination of antimicrobial agents in bone.

A microtechnique, requiring very small amounts of tissue material, was developed for assay of antimicrobial agents in bone. Without previous homogenization or extraction, small bone pieces (mean weight 0.014 g) from human subjects and pigs were placed into wells in agar plates preinoculated with the test strain. Round and distinct zones of inhibition were formed around the pieces. Standards for ampicillin and flucloxacillin were prepared from freeze-dried bone pieces from human subjects and pigs with known amounts of antibiotics as well as in human plasma and phosphate-buffered saline (PBS). Curves obtained from these standards were linear. Bone pieces from human and pig maxilla gave superimposable curves, but differed from curves obtained in plasma or PBS. The method was used in a pharmacokinetic study of bacampicillin in human maxillary bone and plasma. Bacampicillin tablets (2 X 400 mg) were given to patients before oral surgery. Standardized bone pieces and plasma samples were obtained at different times during surgery. The peak ampicillin concentrations estimated from the population curves were 8.0 mg/l in plasma and 1.1 mg/l in maxillary bone. The elimination half-life of ampicillin was similar in plasma and maxillary bone.

Comparative bioavailability and half-lives of cloxacillin and flucloxacillin.

The bioavailability of cloxacillin and flucloxacillin were compared after the oral administration, in capsule form, of 500 mg cloxacillin or 250 mg flucloxacillin to healthy volunteers. There were no significant differences observed in time to peak concentrations, peak concentrations or area under the serum concentration/time curve. However, flucloxacillin had a significantly longer serum elimination half-life. Serum levels of cloxacillin and flucloxacillin were determined using a high-performance liquid chromatographic assay.

The treatment of infections in general practice with magnapen: a multicentre trial.

This study was carried out in general practice. Six hundred and forty-seven infections of the respiratory tract, urinary tract and the skin and soft tissues in 646 patients were treated with oral Magnapen (250 mg ampicillin + 250 mg flucloxacillin) four times each day. An overall success rate of 94% was achieved. The main side-effect was diarrhoea causing 2.5% of patients to discontinue therapy. These results demonstrate the outstanding value of Magnapen in treating a wide variety of infections in general practice.