Prevalence of antimicrobial resistance among bacterial pathogens isolated from cattle in different European countries: 2002–2004

Background

The project "Antibiotic resistance in bacteria of animal origin – II" (ARBAO-II) was funded by the European Union (FAIR5-QLK2-2002-01146) for the period 2003–2005, with the aim to establish a continuous monitoring of antimicrobial susceptibility among veterinary laboratories in European countries based on validated and harmonised methodologies. Available summary data of the susceptibility testing of the bacterial pathogens from the different laboratories were collected.

Method

Antimicrobial susceptibility data for several bovine pathogens were obtained over a three year period (2002–2004). Each year the participating laboratories were requested to fill in excel-file templates with national summary data on the occurrence of antimicrobial resistance from different bacterial species.A proficiency test (EQAS – external quality assurance system) for antimicrobial susceptibility testing was conducted each year to test the accuracy of antimicrobial susceptibility testing in the participating laboratories. The data from this testing demonstrated that for the species included in the EQAS the results are comparable between countries.

Results

Data from 25,241 isolates were collected from 13 European countries. For Staphylococcus aureus from bovine mastitis major differences were apparent in the occurrence of resistance between countries and between the different antimicrobial agents tested. The highest frequency of resistance was observed for penicillin. For Mannheimia haemolytica resistance to ampicillin, tetracycline and trimethoprim/sulphonamide were observed in France, the Netherlands and Portugal. All isolates of Pasteurella multocida isolated in Finland and most of those from Denmark, England (and Wales), Italy and Sweden were susceptible to the majority of the antimicrobials. Streptococcus dysgalactiae and Streptococcus uberis isolates from Sweden were fully susceptible. For the other countries some resistance was observed to tetracycline, gentamicin and erythromycin. More resistance and variation of the resistance levels between countries were observed for Escherichia coli compared to the other bacterial species investigated.

Conclusion

In general, isolates from Denmark, England (and Wales), the Netherlands, Norway, Sweden and Switzerland showed low frequencies of resistance, whereas many isolates from Belgium, France, Italy, Latvia and Spain were resistant to most antimicrobials tested. In the future, data on the prevalence of resistance should be used to develop guidelines for appropriate antimicrobial use in veterinary medicine.     

In vitro antimicrobial activity of hydroxy and N4-acetyl sulphonamide metabolites

Hydroxylated metabolites of sulphadimidine, sulphamerazine, sulphatroxazole, sulphamethoxazole, and sulphadiazine exhibited antimicrobial activity against Escherichia coli 28 PR 271 test strain ranging from 2.5 to 39.5 per cent of that of the parent drug. Trimethoprim addition potentiated the antimicrobial activity of these metabolites. N4-acetyl sulphonamide metabolites possessed no antimicrobial activity, nor did trimethoprim potentiated them.     

In vitro antimicrobial activity of hydroxy and N4‐acetyl sulphonamide metabolites

Summary

Hydroxylated metabolites of sulphadimidine, sulphamerazine, sulphatroxazole, sulphamethoxazole, and sulphadiazine exhibited antimicrobial activity against Escheria coli 28 PR 271 test strain ranging from 2.5 to 39.5 per cent of that of the parent drug. Trimethoprim addition potentiated the antimicrobial activity of these metabolites. N₄‐acetyl sulphonamide metabolites possessed no antimicrobial activity, nor did trimethoprim potentiated them.     

Antimicrobial Resistance of Faecal Escherichia coli Isolates from Pig Farms with Different Durations of In‐feed Antimicrobial Use

Antimicrobial use and resistance in animal and food production are of concern to public health. The primary aims of this study were to determine the frequency of resistance to 12 antimicrobials in Escherichia coli isolates from 39 pig farms and to identify patterns of antimicrobial use on these farms. Further aims were to determine whether a categorization of farms based on the duration of in‐feed antimicrobial use (long‐term versus short‐term) could predict the occurrence of resistance on these farms and to identify the usage of specific antimicrobial drugs associated with the occurrence of resistance. Escherichia coli were isolated from all production stages on these farms; susceptibility testing was carried out against a panel of antimicrobials. Antimicrobial prescribing data were collected, and farms were categorized as long term or short term based on these. Resistance frequencies and antimicrobial use were tabulated. Logistic regression models of resistance to each antimicrobial were constructed with stage of production, duration of antimicrobial use and the use of 5 antimicrobial classes included as explanatory variables in each model. The greatest frequencies of resistance were observed to tetracycline, trimethoprim/sulphamethoxazole and streptomycin with the highest levels of resistance observed in isolates from first‐stage weaned pigs. Differences in the types of antimicrobial drugs used were noted between long‐term and short‐term use farms. Categorization of farms as long‐ or short‐term use was sufficient to predict the likely occurrence of resistance to 3 antimicrobial classes and could provide an aid in the control of resistance in the food chain. Stage of production was a significant predictor variable in all models of resistance constructed and did not solely reflect antimicrobial use at each stage. Cross‐selection and co‐selection for resistance was evident in the models constructed, and the use of trimethoprim/sulphonamide drugs in particular was associated with the occurrence of resistance to other antimicrobials.     

Minimal inhibitory concentrations of antimicrobial agents against Actinobacillus pleuropneumoniae.

Forty-five isolates of Actinobacillus pleuropneumoniae were tested for susceptibility to 12 antimicrobial agents using a microdilution method for the minimal inhibitory concentration determinations. These results confirmed the high prevalence of A. pleuropneumoniae strains resistant to antibiotics as reported earlier using the disc diffusion method (Kirby-Bauer method). While 36% of the isolates were resistant to the penicillins, 47% were resistant to chloramphenicol and 68% were resistant to tetracycline. Minimal inhibitory concentrations for the resistant isolates were approximately 32 times higher than those for the susceptible isolates to the above antibacterial agents. The isolates were in general weakly susceptible or resistant to spectinomycin, lincomycin, tiamulin and spiramycin whereas most of them were susceptible to gentamicin, trimethoprim and erythromycin. The susceptibility pattern was similar throughout the 1980 to 1984 period. The 14 serotype 5 isolates were more resistant to tetracycline but less resistant to chloramphenicol and the penicillins than the 28 serotype 1 isolates.     

In vitro antibacterial susceptibility of Arcobacter butzleri isolated from different sources

The objective of this study was to determine the antibiotic susceptibility of Arcobacter spp. isolated from various sources. Seventy Arcobacter spp. isolates were tested for their susceptibility to 13 antimicrobial agents. Antimicrobial susceptibility testing was performed by using the agar disc diffusion method on Mueller-Hinton agar supplemented with 5% defibrinated sheep blood. The antibiotics tested included enrofloxacin, erythromycin, streptomycin, gentamycin, rifampin, tetracycline, ampicillin, trimethoprim/sulfamethoxazole, nalidixic acid, danofloxacin, amoxycillin-clavulonic acid, cefuroxime-sodium, neomycine. Although all the arcobacters tested were susceptible to gentamycin, resistance to three or more antibacterial agents (especially, trimethoprim/sulfamethoxazole, cefuroxime-sodium and rifampin) was observed. A. butzleri isolates were found to be resistant to amoxycillin+clavulonic acid, nalidixic acid and ampicillin, at the rate of 20%, 44.28% and 78.57% respectively. In conclusion, gentamycin, streptomycin and tetracycline may be suitable antibiotics for the treatment or control of disease caused by Arcobacter spp. in veterinary and human medicine.     

In vitro antibiotic sensitivity of strains of Ornithobacterium rhinotracheale isolated in The Netherlands between 1996 and 1999.

All the strains of Ornithobacterium rhinotracheale isolated in the Netherlands between 1996 and 1999 were tested in the agar gel diffusion test for their sensitivity to amoxycillin, tetracycline, enrofloxacin and trimethoprim/sulphonamide (TMP/S). The percentages of strains sensitive to amoxycillin and tetracycline decreased in successive years from approximately 62 per cent to 14 per cent, and four strains were resistant to enrofloxacin or TMP/S. Twelve multiresistant strains were tested against seven alternative antibiotics; they were resistant to all of them except clavulanic acid-potentiated amoxycillin.     

犬脓皮病病原菌的分离鉴定及耐药性分析

为了研究犬脓皮病致病菌的种类及其对抗生素的敏感性,采用细菌培养、分离、革兰染色和生化试验等方法鉴别病原菌;采用Kirby-Bauer药物纸片琼脂扩散法对病原菌进行药敏试验。结果表明：病犬发病部位的病原菌为中间型葡萄球菌和金黄色葡萄球菌。中间型葡萄球菌对头孢拉啶和头孢氨苄敏感,敏感率为20%;对羧苄西林和头孢他啶中度敏感,中度敏感率为20%;对红霉素、卡那霉素、复方新诺明、哌拉西林、青霉素和苯唑西林产生耐药,耐药率为60%。金黄色葡萄球菌对头孢拉啶和头孢氨苄敏感,敏感率为20%;对羧苄西林和哌拉西林中度敏感,中度敏感率为20%;对红霉素、卡那霉素、复方新诺明、头孢他啶、青霉素和苯唑西林产生耐药,耐药率为60%。     

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.     

Layout proposal for a microtitre plate to be used in routine antimicrobial susceptibility testing of bacteria from infections of dogs and cats

The determination of minimum inhibitory concentrations by broth microdilution is recommended as method of choice for susceptibility testing of veterinary bacterial pathogens. Accordingly, broth microdilution is used in veterinary routine diagnostic laboratories at a progressive rate. To reduce the costs of susceptibility testing, it is reasonable to develop widely accepted uniform microtitre plate layouts that are produced in large quantities. Such microtitre plate layouts have already been developed and published for the susceptibility testing of pathogens from food-producing animals. However, a microtitre plate layout, especially designed for the testing of bacteria from dogs and cats, should be available, too. The choice of the antimicrobial agents or combinations of antimicrobial agents to be included in a suitable layout should be based on the following criteria: (1) the approval and availability of an antimicrobial agent or combination of agents, (2) known cross-resistances, and (3) availability of approved clinical breakpoints. The latter point is of particular importance for the choice of the numbers of concentrations per antimicrobial agent tested and the range of test concentrations. Taking into account these aspects, a science-based layout proposal for microtitre plates, which are suitable for routine testing of bacteria from dogs and cats, is presented and discussed.     

Antimicrobial susceptibility of Pasteurella multocida and Bordetella bronchiseptica from dogs and cats as determined in the BfT-GermVet monitoring program 2004-2006

A total of 92 canine/feline Pasteurella multocida strains form respiratory tract infections or infections of skin/ear/mouth as well as 42 canine/feline Bordetella bronchiseptica strains from respiratory tract infections were investigated for their susceptibility to antimicrobial agents. While the P. multocida strains were susceptible to all antimicrobial agents tested - except sulfonamides -, a considerable number of the B. bronchiseptica strains was resistant or exhibited high MIC values against a number of antimicrobial agents including penicillin G, oxacillin, cefazolin, ceftiofur, cefquinome, sulfamethoxazole, and trimethoprim/sulfamethoxazole.     

Antimicrobial susceptibility of Mycoplasma hyorhinis

A broth microdilution technique was used to determine the antimicrobial susceptibility of 15 field isolates of Mycoplasma hyorhinis to 10 antimicrobial agents, representative of different classes, and contrasting newer agents to existing ones. For the macrolides, the MIC(90) for tylosin and tilmicosin was 1 and 4 microg/ml, respectively, but was > or = 16 microg/ml for erythromycin. Tetracycline, lincomycin and enrofloxacin each had an MIC(90) of 2 microg/ml. The mycoplasma had similar levels of susceptibility to the aminoglycoside and aminocyclictol classes exhibiting an MIC(90) of 4 microg/ml for gentamicin and 2 microg/ml for spectinomycin. The isolates exhibited high MICs to trimethoprim/sulfamethoxazole with an MIC(90) > or = 16/304 microg/ml. In summary, M. hyorhinis isolates from the US had low MICs against a variety of antimicrobials tested, with the exception of erythromycin and trimethoprim/sulfamethoxazole.     

Results from a German interlaboratory test to establish the broth microdilution method for the determination of the minimum inhibitory concentration of bacteria from animals

In accordance with NCCLS guideline M31-A2, the DVG working group "antimicrobial resistance" developed a standard operating procedure (SOP) for the determination of the minimal inhibitory concentration (MIC) of antimicrobial agents by broth microdilution. This SOP was evaluated for its fitness for use in a national interlaboratory test. A total of 32 participating laboratories tested five strains (including two internationally accepted reference strains and three field strains representing in total three different bacterial species) three times at a one week interval each, using uniform microtitre plates. In 31 of the 32 laboratories more than 80% of MIC determinations performed yielded values in the expected range. In total 94.0% of the results were reproducible, with a lesser deviation of 4.0% from the expected values for laboratories performing MIC determination as a matter of routine (46.9%), compared to 7.9% for laboratories without such routine (53.1%). Comparing the consistency of results on the basis of the tested strains, a higher reproducibility of the results was observed for reference strains (96.1%) than for field strains (92.6%). In particular results obtained for the Streptococcus uberis field strain were afflicted with a higher error ratio (98 deviations from the expected values). Among the tested antimicrobial agents, a higher variability of results was recorded only for gentamicin with 16.7% divergent MIC determinations (mean value 6.0%). The high reproducibility of the results confirmed by this interlaboratory study underlines the robustness of the developed SOP as well as broth microdilutions as the method of choice for MIC determina tion.     

Quantitative susceptibility of Streptococcus suis strains isolated from diseased pigs in seven European countries to antimicrobial agents licensed in veterinary medicine

The susceptibility of Streptococcus suis strains (n=384) isolated from diseased pigs in seven European countries to 10 antimicrobial agents was determined. For that purpose a microbroth dilution method was used according to CLSI recommendations. The following antimicrobial agents were tested: ceftiofur, cefquinome, enrofloxacin, florfenicol, gentamicin, penicillin, spectinomycin, tetracycline, tilmicosin and trimethoprim/sulphamethoxazole. Using breakpoints established by CLSI for veterinary pathogens, all strains were susceptible to ceftiofur, florfenicol, enrofloxacin and penicillin. MIC-90 values of these antibiotics were < or = 0.03, 0.5, 2 and < or = 0.13 microg/mL, respectively. A low degree of resistance was observed for gentamicin (1.3%), spectinomycin (3.6%) and trimethoprim/sulphamethoxazole (6.0%). MIC-90 values of these antibiotics were 8, 16 and 2 microg/mL, respectively. A high level of resistance was observed for tetracycline (75.1%). A MIC-90 value of 64 microg/mL was found for this antibiotic. Serotype-associated differences in MIC-90 values were observed for tetracycline, tilmicosin and trimethoprim/suphamethoxazole.     

鸡源印第安纳沙门菌多重耐药性分析

采集山东不同地区鸡源沙门菌,根据Kauffmann-White方法测定分离株血清型,采用肉汤微量稀释法测试分离株对16种抗菌药物的敏感性,PCR方法检测10种耐药基因,分析耐药表型和耐药基因之间的关系。结果显示：共鉴定出沙门菌80株,其中印第安纳沙门菌60株。药敏试验证实：60株印第安纳沙门菌对阿莫西林-克拉维酸、头孢唑啉、多黏菌素、氨苄西林、多西环素和甲氧苄啶等16种抗菌药物普遍耐药。88.33%菌株多重耐药分布在12~15耐,未发现3耐以下的菌株。PCR扩增出int1,blaTEM,aac（6’）-Ib-cr,floR,catA1,tetA,strA,cmlA 8种耐药基因。超过90%的菌株携带int1,blaTEM,floR和aac（6’）-Ib-cr耐药基因。上述结果表明,耐药表型及耐药基因的符合呈现相关性。     

Prevalence and antimicrobial resistance of Salmonella spp. in non-diarrhoeic dogs in Trinidad

The estimated prevalence and antimicrobial resistances of Salmonella spp. in non-diarrhoeic dogs across Trinidad was determined. The serotypes of Salmonella spp. isolated were also identified. Of a total of 1391 dogs sampled, 50 (3.6%) were positive for Salmonella spp. with 28 different serotypes, the predominant serotypes were Javiana (12), Newport (6), Arechavaleta (5) and Heidelberg (5). Fifty-seven (85.1%) of 67 isolates exhibited resistance to one or more antimicrobial agents. Of eight antimicrobial agents tested, resistance was exhibited to streptomycin (80.6%), cephalothin (37.3%), neomycin (38.8%) and gentamicin (9.0%). All isolates were sensitive to ampicillin, norfloxacin, choramphenicol and sulphamethoxazole/trimethoprim. It was concluded that the isolation of the Salmonella spp. from non-diarrhoeic dogs could pose health hazard to their owners as most serotypes are known to be virulent. Furthermore, the prevalence of resistance to antimicrobial agents amongst the Salmonella isolates from these animals indicates susceptibility testing may influence chemotherapeutic choices when treating these isolates.     

Prevalence of antimicrobial resistance among Salmonella on midwest and northeast USA dairy farms

The objective of this study was to determine the prevalence of antimicrobial resistance among Salmonella isolated from dairy herds in New York, Minnesota, Michigan, and Wisconsin, USA. Serogroup and antimicrobial susceptibility characteristics were determined for Salmonella from cattle and environmental samples collected during August 2000–October 2001 as part of a longitudinal study where 129 herds were visited at 2-month intervals. Salmonella isolates were tested (using a broth microdilution method) for susceptibility to amoxicillin/clavulanic acid, ampicillin, ceftiofur, ceftriaxone, cephalothin, chloramphenicol, ciprofloxacin, gentamicin, kanamycin, nalidixic acid, streptomycin, sulfamethoxazole, tetracycline, and trimethoprim/sulfamethoxazole. Of the 1506 isolates tested for minimum inhibitory concentrations to these 14 antimicrobial agents, 81.2% were pan-susceptible and for most herds (81.6%) the predominant antimicrobial resistance pattern was pan-susceptible. At least 1 Salmonella isolate resistant to 5 or more antimicrobial agents was found on 23.6% of herds. This resistance phenotype was most common among serogroups B and E1 and among samples from calves and farmer-designated sick cows. Resistant samples most frequently exhibited resistance to tetracycline, streptomycin, and/or ampicillin. No samples were resistant to ceftriaxone (though 13 were in the intermediate range), and very few samples were resistant to ciprofloxacin (n = 1), nalidixic acid (n = 5), or trimethoprim/sulfamethoxazole (n = 7).     

In vitro sensitivity of Hungarian Actinobaculum suis strains to selected antimicrobials

In vitro antimicrobial sensitivity of 12 Hungarian isolates and the type strain ATCC 33144 of Actinobaculum suis to different antimicrobial compounds was determined both by the agar dilution and by the disc diffusion method. By agar dilution, MIC50 values in the range of 0.05-3.125 micrograms/ml were determined for penicillin, ampicillin, ceftiofur, doxycycline, tylosin, pleuromutilins, chloramphenicol, florfenicol, enrofloxacin and lincomycin. The MIC50 value of oxytetracycline and spectinomycin was 6.25 and 12.5 micrograms/ml, respectively. For ofloxacin, flumequine, neomycin, streptomycin, gentamicin, nalidixic acid, nitrofurantoin and sulphamethoxazole + trimethoprim MIC50 values were in the range of 25-100 micrograms/ml. With the disc diffusion method, all strains were sensitive to penicillin, cephalosporins examined, chloramphenicol and florfenicol, tetracyclines examined, pleuromutilins, lincomycin and tylosin. Variable sensitivity was observed for fluoroquinolones (flumequine, enrofloxacin, ofloxacin), most of the strains were susceptible to marbofloxacin. Almost all strains were resistant to aminoglycosides but most of them were sensitive to spectinomycin. A strong correlation was determined for disc diffusion and MIC results (Spearman's rho 0.789, p < 0001). MIC values of the type strain and MIC50 values of other tested strains did not differ significantly. Few strains showed a partially distinct resistance pattern for erythromycin, lincomycin and ampicillin in both methods.     

Prevalence and Antimicrobial Resistance of Salmonella spp. in Non‐diarrhoeic Dogs in Trinidad

The estimated prevalence and antimicrobial resistances of Salmonella spp. in non‐diarrhoeic dogs across Trinidad was determined. The serotypes of Salmonella spp. isolated were also identified. Of a total of 1391 dogs sampled, 50 (3.6%) were positive for Salmonella spp. with 28 different serotypes, the predominant serotypes were Javiana (12), Newport (6), Arechavaleta (5) and Heidelberg (5). Fifty‐seven (85.1%) of 67 isolates exhibited resistance to one or more antimicrobial agents. Of eight antimicrobial agents tested, resistance was exhibited to streptomycin (80.6%), cephalothin (37.3%), neomycin (38.8%) and gentamicin (9.0%). All isolates were sensitive to ampicillin, norfloxacin, choramphenicol and sulphamethoxazole/trimethoprim. It was concluded that the isolation of the Salmonella spp. from non‐diarrhoeic dogs could pose health hazard to their owners as most serotypes are known to be virulent. Furthermore, the prevalence of resistance to antimicrobial agents amongst the Salmonella isolates from these animals indicates susceptibility testing may influence chemotherapeutic choices when treating these isolates.