Comparison of Different Agar Diffusion Methods for the Detection of Antimicrobial Residues in Slaughter Animals

The different agar diffusion methods were compared using antibiotic and sulphonamide-impregnated filter-paper discs and the kidneys of healthy and emergency-slaughtered pigs and cows after slaughter.No method used seemed to be sensitive to all antimicrobial drugs preimpregnated onto discs. Tetracycline yielded a greater zone of inhibition at pH 6 than at pH 8 and aminoglycosides, erythromycin, polymyxin B and lin cornycin at pH 8 than at pH 6. It therefore seems necessary to use different pHs (6 and 8). The addition of trimethoprim to the medium is necessary for the detection of sulphonamides. Bacillus subtilis BGA used as the test organism was more sensitive to sulphonamides on the “Test agar for the inhibitor test” containing trimethoprim than on the “Iso-Sensitest agar” also containing trimethoprim. The addition of trimethoprim to “Test agar for the inhibitor test” is recommended at pH 8 but not at pH 6 because false-positive cases (with inhibition zones > 2 mm) were observed at pH 6 with trimethoprim on the kidneys of healthy pigs.     

Characterisation and antimicrobial sensitivity of haemophili isolated from pigs

A total of 70 haemophili from Australia pigs was compared with a range of reference strains of porcine haemophili. Forty-eight of the isolates were identified as Actinobacillus pleuropneumoniae biovar 1 and the remaining 22 isolates as Haemophilus parasuis. Forty one of the A. pleuropneumoniae isolates were used in a study to determine to the minimal inhibitory concentration (MIC) of 12 antimicrobial agents, or combinations of agents. Penicillin, neomycin, trimethoprim, trimethoprim-sulphamethoxazole and tetracycline all showed low MIC values, indicating their potential for the treatment of porcine pleuropneumonia, although 2 isolates showed resistance to tetracycline. A wide range of MIC values was encountered with the sulphonamides.     

Some pharmacokinetic aspects of four sulphonamides and trimethoprim, and their therapeutic efficacy in experimental Escherichia coli infection in poultry

Pharmacokinetic studies in broilers and layers of different sulphonamides indicate a good absorption and a long elimination half-life (of sulphaquinoxaline, sulphadimidine and to a lesser degree sulphadiazine) resulting in high plasma concentrations during drinking water medication in the recommended therapeutic doses. In contrast drinking water medication with high concentrations of trimethoprim (up to 1,320 mg/liter) resulted in a maximal mean plasma concentration of 1.2 micrograms/ml. Very good therapeutic effects were demonstrated in broilers experimentally infected with a sulphonamide-susceptible E. coli strain when treated with sulphaquinoxaline (200 mg/liter), sulphadimidine sodium (2 gram/liter), sulphachloropyridazine 30 per cent (1 gram/liter) and to a lesser degree sulphadiazine sodium (250 mg/liter). Synergism was demonstrated between trimethoprim and sulphadiazine (1:5). The combination of trimethoprim with sulphaquinoxaline (1:3) did not induce better therapeutic effects than sulphaquinoxaline in proportional doses. However, significant synergism was demonstrated between trimethoprim and both sulphonamides in treatment of experimental infection with sulphonamide-resistant E. coli. No signs resembling sulphonamide intoxication were observed during these studies.     

Absorption of two trimethoprim/sulphonamide combinations from the uterus of pony mares

Plasma drug concentrations were measured after two commercially available potentiated sulphonamides, trimethoprim and sulfadoxine and trimethoprim and sulphadiazine, were infused daily for 2 and 3 days, respectively, into the uteri of pony mares which had been mated before ovulation. Intravenous administration of trimethoprim and sulfadoxine allowed uterine absorption of trimethoprim (23-43%) and sulfadoxine (29-34%) to be calculated. After intra-uterine administration trimethoprim and sulphadiazine were detected in the milk of a lactating mare. In order to maintain plasma concentrations likely to be required for clinical efficacy of both drugs they should be administered every 12 h. However, infusions of both preparations caused endometrial inflammation as assessed by cytological and histological examination and this may have been responsible for the low pregnancy rate.     

Antibiotic Resistance of Vibrio anguillarum,in Relation to Serovar and Plasmid Contents

A total of 520 Vibrio anguillarum strains, isolated from fish and the environment, were tested for their sensitivity to 20 different antibiotics. Most isolates were of European origin. The results were compared with data on the O-serogroup and plasmid contents. All strains were sensitive to neomycin, spectinomycin, nitrofurantoin, flumequine and oxolinic acid, while most strains were sensitive to streptomycin, Oxytetracycline, chloramphenicol, sulphonamides, trimethoprim, sulphonamides with trimethoprim, nalidixan, rifampicin, novobiocin and O/129. A major part of the strains were resistant to the macrolides, spiramycin and lincomycin. For ampicillin, cephalothin, and Colistin marked differences were recorded with respect to O-serogroup. Most O1 strains were resistant to Colistin and sensitive to ampicillin and cephalothin, while most O2 strains were sensitive to Colistin but resistant to ampicillin and cephalothin. Some antibiotic resistant strains carried plasmids but no conjugation experiments were carried out to detect possible R factors.     

Evaluation of a mixture of trimethoprim and sulphaquinoxaline for the treatment of bacterial and coccidial diseases of poultry

Representative experiments from work undertaken to develop a synergistic mixture of trimethoprim and sulphaquinoxaline for the preventive treatment of certain poultry diseases are described. Sulphaquinoxaline in the diet for four days was shown to achieve at least an 85 per cent higher blood level than nine other sulphonamides in chicks, and the efficacies of various trimethoprim/sulphaquinoxaline regimes in the diet or in the drinking water were demonstrated against pasteurellosis, colisepticaemia and five kinds of coccidiosis. Regimes for bacterial diseases were begun one day before infection but those for coccidial diseases were begun on the same day as infection or later. Overall, a total dose of 30 mg/kg bodyweight/day (trimethoprim/sulphaquinoxaline = 1:3) controlled these seven diseases. The same treatment was also shown to control sulphaquinoxaline-resistant strains of Escherichia coli and Eimeria acervulina. Although both drinking water and food were used for drug administration, twice the inclusion rate was required in food to that in water for equivalent efficacy. The significance of different modes of expression of dosages for bacterial and coccidial diseases is explained.     

Salmonella enteritis in dogs,not relevant?

In order to shed some light on the different opinions regarding the importance of Salmonella infections in dogs, we gathered some data from our laboratory database. Of the 6589 faecal samples from diarrhoeic dogs examined, 69 (1%) yielded Salmonella spp. Another eleven isolates were cultured from materials other than faeces. If Salmonella spp. can be cultured directly from faeces, this should be interpreted as clinically significant; however, even if the organism is found only after enrichment it may still be a clinical case. Antibiotic therapy is indicated in all dogs that are severely ill or have systemic infection, fluoroquinolones or trimethoprim/sulphonamides being drugs of choice. Double-blind placebo-controlled studies of humans with gastroenteritis have shown that the length of postconvalescent excretion of Salmonella is not affected by antimicrobials. For the elimination of the carrier state, however, fluoroquinolones are successful in human medicine. Salmonellae are zoonotic agents and transmission from dog to man has been reported. This should be taken into account when deciding on antimicrobial therapy. Of our isolates (n = 80), 96% were susceptible to enrofloxacin, 94% to trimethoprim/sulphonamides, 94% to gentamicin, 86% to ampicillin and amoxicillin-clavulanic acid, 47% to cephalexin and 63% to tetracycline.     

Antibacterial drugs used against mastitis in cattle by the systemic route

The veterinarian in clinical practice is often confronted with cases of mastitis that require systemic antibacterial treatment in addition to local treatment. This paper reviews the suitability of drugs available in New Zealand, taking into account their anti-staphylococcal activity, routes of administration, and their ability to attain and maintain therapeutic levels in the udder following systemic administration. The drugs considered include the more common penicillins, aminoglycosides and macrolides; oxytetracyline, chloramphenicol, trimethoprim, and several sulphonamides.

The success of systemic therapy against mastitis depends to a large extent on the concentration of antibacterial drug achieved at foci of infection. Passage of drugs across the blood-milk barrier takes place by passive diffusion, and the factors influencing this diffusion are discussed.

Whe mastitis is associated with sensitive organisms, penicillin is recommended, although, as with all other antibacterials discussed, the dose used must be higher than normal. For penicillin, doses of the order of 16,500 iu/kg are recommended.

The intramuscular injection of oxytetracycline will not produce therapeutic levels in milk but, after intravenous injection of high doses (10 mg/kg), this antibiotic is likely to maintain therapeutic levels in milk over a 24-hour period.

As a first choice for the systemic treatment of mastitis, either tylosin or erythromycin is recommended. At a dose rate of 12.5 mg/kg, either will maintain for 24 hours milk levels in excess of the average MICs for staphylococci.

Of the sulphonamides, sulphanilamide and sulphadimidine produce the highest milk levels. After intravenous administration at a dose rate of 200 mg/kg, sulphadimidine will maintain therapeutic levels in milk for 12 hours.

Although trimethoprim has a very short half-life in cattle which limits its usefulness, it readily enters the milk and a combination of trimethoprim (as a suspension) with sulphadiazine, at a dose rate of 48 mg/kg, might be expected to maintain therapeutic levels in the milk for 12 hours.

It is concluded that dihydrostreptomycin, neomycin, chloramphenicol, and the combination sulphadoxine/trimethoprim, are not suitable for the systemic treatment of bovine mastitis.     

Tissue damage and concentration at the injection site after intramuscular injection of chemotherapeutics and vehicles in pigs.

Intramuscular injection sites were examined for macroscopical and microscopical changes and for residues of drugs six and 30 days after injection of chemotherapeutic preparations or vehicles in swine. The chemotherapeutic preparations contained sulphonamide and/or trimethoprim. All the chemotherapeutic preparations and the vehicles except physiological saline and sterile water caused macroscopical and microscopical changes, mainly appearing as areas of necrotic muscle tissue six days after the injection and as scar tissue 30 days after the injection. Residues of drugs were found at nearly all the injection sites six days after the injection, while 30 days after the injection only residues of sulphonamides were detectable in nearly half of the injection sites.     

Susceptibility of Pseudomonas pseudomallei isolates of non-human origin to chemotherapeutic agents by the single disc sensitivity method

Single disc sensitivity tests against 42 strains of Pseudomonas pseudomallei isolated from soil, water, sheep, pigs, goats, birds, a horse and a tree kangaroo have shown that a remarkably similar pattern occurs to that obtained in isolates from human cases. Generally, the organisms are sensitive to kanamycin, novobiocin, compound sulphonamides, sulphamethoxazole/trimethoprim and tetracycline and resistant to ampicillin, bacitracin, cloxacillin, colistin, erythromycin, gentamycin, nitrofurantoin, penicillin, polymyxin B, streptomycin and vancomycin. Sensitivity to neomycin was variable.     

Usage of antimicrobials and occurrence of antimicrobial resistance among bacteria from mink

The usage of antimicrobials for treatment of mink and the occurrence of antimicrobial resistance among the most important bacterial pathogens in mink was investigated. The aim of the study was to provide data, which may serve as a basis for the formulation of recommendations for prudent use of antimicrobials for mink. A total of 164 haemolytic staphylococci, 49 haemolytic streptococci, 39 Pseudomonas aeruginosa, 13 Pasteurella multocida, and 1093 Escherichia coli isolates from Danish mink were included in the study. A high frequency of resistance among S. intermedius was found for tetracyclines (54.7%), followed by penicillin (21.7%), lincosamides (20.4%), macrolides (19.1%), and spectinomycin (18.5%). Very low frequencies of resistance were recorded for other antimicrobials. The highest frequency among the E. coli isolates was recorded for ampicillin, streptomycin, sulphonamides, and tetracyclines, whereas resistance to other antimicrobials was rare. All P. aeruginosa were sensitive to gentamicin and colistin and sensitive or intermediate to enrofloxacin, whereas most isolates were resistant to all other antimicrobials. All P. multocida and haemolytic streptococci were sensitive to penicillin. There was a steady increase in the use of antimicrobials during the period 2001-2006, the majority of the prescribed amount being extended spectrum penicillins followed by aminoglycosides, sulphonamides with trimethoprim, and macrolides.     

Aspects of bacterial resistance to antimicrobials used in veterinary dermatological practice

Aspects of bacterial resistance to the major classes of antimicrobials used in veterinary dermatology are presented in this review. Resistance of gram-positive and gram-negative bacteria to tetracyclines, macrolide-lincosamide-streptogramin antibiotics, chloramphenicol, mupirocin, sulphonamides, trimethoprim, aminoglycosides, fluoroquinolones and β-lactam antibiotics are depicted with respect to the different mechanisms of acquired and intrinsic resistance. Examples are given for the three major resistance mechanisms, enzymatic inactivation, decreased intracellular drug accumulation and target modification. In addition, basic information about mobile genetic elements which carry resistance genes, such as plasmids, transposons and gene cassettes, and their modes of spreading via transduction, conjugation, mobilization and transformation is provided.     

高效液相色谱—紫外法对13种磺胺类药物的同步检测

本研究旨在建立同步测定多种磺胺类药物残留的高效液相色谱—紫外法。通过对磺胺嘧啶(SD)、磺胺醋酰(SA)、磺胺喹噁啉(SQ)、磺胺氯丙嗪(SUL)、磺胺间甲氧嘧啶(SMM)、磺胺对甲氧嘧啶(SMD)、磺胺二甲氧嘧啶(SDM')、磺胺异噁唑(SIZ)、磺胺噻唑(ST)、磺胺噻唑钠(ST-Na)、酞磺胺噻唑(PST)、磺胺甲噁唑(SMZ)、琥珀酰磺胺噻唑(SST)等13种磺胺类药物进行紫外波长扫描分析,选择色谱检测的波长为270 nm。由于磺胺类药物的酸碱度不同及结构差异,在经过多种组合比较后,选择乙腈和乙酸水溶液作为流动相进行梯度洗脱,建立的条件可将这些药物分开,13种药物可以分离得到10个色谱峰,ST-Na、PST和ST 3种成分重叠,SDM'和SMZ重叠。用乙腈—氯仿作样品提取并抽提,杂质干扰较少,回收率80%以上,分析用时相对较短。     

Characterization of Streptococcus suis serotype 7 isolates from diseased pigs in Denmark

Isolates of Streptococcus suis serotype 7 from diseased pigs in Denmark were characterized by ribotyping, pulsed field gel electrophoresis (PFGE), MIC-determinations and detection of resistance genes. Forty-one different ribotype profiles were found among the 103 isolates and could be divided into two main clusters. No obvious relationship between ribotypes and the clinical origin of the isolates could be observed. Fifty-four isolates, including all 24 isolates belonging to the main ribotype profile were examined by PFGE and 50 different profiles were found. A high frequency of resistance to erythromycin (41%), tetracycline (24%) and streptomycin (28%) was observed. Furthermore, almost all isolates (101) were resistant to sulphamethoxazol. Most isolates were susceptible to ceftiofur, chloramphenicol, florfenicol, penicillin, ciprofloxacin, trimethoprim and trimethoprim + sulphonamides. The tet(M) gene was found among 11 (44%) and the tet(O) gene in six (24%) of 25 tetracycline resistant isolates. The tet(L) and tet(S) genes were not detected in any isolates. The erm(B) gene was detected in 39 (93%) of 42 erythromycin resistant isolates.     

Antimicrobial residues in milk. Comparison of different agar diffusion methods

Different agar diffusion methods were compared in order to find a sensitive method for the detection of various antimicrobial residues in milk. A total of 588 producer milk samples were analyzed using subsets of the most sensitive methods.With the IDF method, 2 positive cases (0.34 %) appeared among the producer milk samples, with the Thermocult method 13 positive cases (2.21 %) and with the Test agar pH 8 method with trimethoprim and glucose 4 positive cases (0.68 %). A combination of the IDF method and the Test agar pH 8 method resulted in 6 positive cases (1.02 %) and a combination of the Thermocult method and the Test agar pH 8 method in 17 positive cases (2.89 %). With penicillinase 41 % of the positive cases were identified as β-lactam antibiotics and with p-aminobenzoic acid 18 % of the positive cases were identified as sulphonamides. 41 % of the positive cases remained unexplained.The best combination for the detection of antimicrobial agents in milk seems to be that of the Thermocult method and the Test agar pH 8 method with trimethoprim and glucose.     

Tetracycline-resistance in lactose-positive enteric coliforms originating from Belgian fattening pigs: degree of resistance, multiple resistance and risk factors

Between March and October 2003 a field study was conducted in 50 randomly selected pig herds to assess the degree of tetracycline-resistance in lactose-positive enteric coliforms (LPEC) originating from fattening pigs and to evaluate the combined effects of various husbandry conditions on the development and persistence of antimicrobial-resistant bacteria.

Data on housing, management and antimicrobial-drug consumption were collected, as well as faecal samples at three production stages: end of the nursery period (mean age: 72 days), end of the grower period (mean age: 125 days) and end of the finisher period (mean age: 186 days). The degree of tetracycline-resistant LPEC was determined by means of an agar dilution method.

Tetracycline-resistant LPEC were found in every herd. The overall degree of tetracycline-resistance in LPEC was 56.8% (S.D. 22.4%). Only a very weak relation was found between the degrees of TETR in the different production stages within the same herd, indicating that the degree of TETR is mainly associated with the production stage rather than with the farm as a whole.

The risk factor analysis showed that besides the antimicrobial-drug use, other factors like inside pen hygiene can influence the development and maintenance of antimicrobial-resistant bacteria in the gastrointestinal tracts of pigs. It was also observed that tetracycline-resistance in commensal Escherichia coli is often linked with resistance to other antimicrobial drugs like ampicillin and trimethoprim–sulphonamides.

These results illustrate that the epidemiology of antimicrobial resistance is influenced by antimicrobial-drug use, cross-resistance development and non-antimicrobial risk factors.     

Baquiloprim, a new antifolate antibacterial: in vitro activity and pharmacokinetic properties in cattle

During examination of the half-lives in cattle of a series of 5-substituted diaminobenzyl-pyrimidines, it was found that replacement of the phenyl ring of trimethoprim (tmp) by bicyclic structures, particularly a quinolyl group, led to increases in half-life. The presence of a dimethylamino group on the quinolyl ring of the compound baquiloprim (bqp) conferred a half-life of about 10 hours. In contrast to tmp (half-life about one hour), bqp was well absorbed from the gastrointestinal tract in all ages of cattle, plasma concentrations reaching a plateau on the day after dosing followed by a slow decline. bqp showed the same high broad spectrum antibacterial activity as tmp, with marked synergy with sulphonamides. Its differential binding of the dihydrofolate reductases of Escherichia coli and rat liver predicted a margin of safety similar to that of tmp. The results of efficacy studies in mice in comparison with tmp showed that the longer half-life of bqp was associated with greater efficacy, and therapeutic properties superior to those of tmp in cattle were therefore predicted for bqp.     

TMP和DVD对两种磺胺药抗球虫增效作用试验

以雏鸡柔嫩艾美耳球虫病为模型,以相对存活率、相对增重率、盲肠病变值和盲肠卵囊值为观察指标,以抗球虫指数(ACI)为判定标准,观察甲氧苄啶(TMP)和二甲氧苄啶(DVD)与不同浓度磺胺喹口恶啉(SQ)和磺胺氯吡嗪钠(Esb3)联合用药治疗效果和增效作用.结果显示,药物对照组0.02%SQ和0.06% Esb3的ACI值分别为151和162;TMP与0.01%、0.02%、0.05%的SQ联用的ACI分别为177、181和195; DVD分别与0.01%、0.02%、0.05%的SQ联用的ACI分别为177、194和194;TMP与0.03%、0.06%、0.12%的Esb3联用的ACI分别为181、188和192;DVD与0.03%、0.06%、0.12%的Esb3联用的ACI分别为185、187和191.结果表明,TMP和DVD能明显提高SQ和Esb3的抗球虫效果, TMP和DVD之间的抗球虫增效作用无明显的差异.     

Susceptibility testing of Actinobacillus pleuropneumoniae in Denmark. Evaluation of three different media of MIC-determinations and tablet diffusion tests

This study was conducted to compare the applicability of three different media in sensitivity testing of Actinobacillus pleuropneumoniae by means of MIC and tablet diffusion tests. The media used were: modified PPLO agar, chocolatized Mueller-Hinton-II and Columbia agar supplemented with NAD. Seven antimicrobial agents were tested: ceftiofur, enrofloxacin, penicillin, spectinomycin, tiamulin, trimethoprim + sulfadiazine and tylosin, against 40 randomly selected A. pleuropneumoniae isolates. In general, good agreement was found between results obtained with all combinations of media, most antimicrobials tested and the two-test systems. Some variations between media were observed for spectinomycin, tiamulin and tylosin. For ceftiofur and trimethoprim + sulfadiazine some isolates with low MIC-values were classified as resistant using tablet diffusion, indicating that the break points of resistance for these antimicrobials using the tablet diffusion tests need adjustment. Using current break points for resistance with MIC-determinations, all isolates tested susceptible to ceftiofur, enrofloxacin, penicillin, tiamulin and trimethoprim + sulfadiazine. A larger number of isolates tested resistant to spectinomycin and tylosin on all three media using both MIC determinations and tablet diffusion.     

Multiple antimicrobial resistant Salmonella enterica serovar Paratyphi B variant Java in cattle: a case report

An epidemiological investigation of a calf rearing premises and a closely associated dairy herd was carried out after the isolation of Salmonella enterica serovar Paratyphi B variant Java phage type 3b variant 2 from clinically diseased calves on the premises. The isolate was resistant to ampicillin, chloramphenicol, streptomycin, sulphonamides, tetracyclines, trimethoprim and cefoperazone. The organism was widespread on the calf unit and was also recovered from the dairy premises, mainly from groups of weaned calves. The investigation was extended to 10 epidemiologically linked farms but no S Java was isolated from any of the 40 to 60 samples collected from each premises. Molecular studies showed that the S Java isolates were genetically most similar to isolates from cases of human disease associated with ornamental fish tanks or feed. Long PCR and resistance gene profiling identified a resistance island which was indistinguishable from the human 'fish tank' strain of S Java and animal and human epidemic strains of S Typhimurium DT104. The isolates were clearly distinguished from multi-resistant S Java strains commonly associated with continental poultry. This is the first report of S Java with this resistance pattern in Great Britain.