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 resistance in staphylococci from animals with particular reference to bovine Staphylococcus aureus, porcine Staphylococcus hyicus, and canine Staphylococcus intermedius.

Besides their role as commensals on the skin and mucosal surfaces, staphylococci may be involved in a wide variety of diseases in animals. Staphylococcal infections in animals are mainly treated with antimicrobial agents and as a consequence, staphylococci from animal sources have developed and/or acquired resistance to the respective antimicrobial agents. Resistance statistics obtained from national monitoring programmes on staphylococci from cattle and pigs, but also from surveillance studies on staphylococci involved in diseases in dogs are reported and reviewed with regard to their comparability. This review mainly focusses on the genetic basis of antimicrobial resistance in staphylococci of animal origin. Particular attention is paid to resistance to those antimicrobial agents which are most frequently used in veterinary medicine, but also to antimicrobial agents, such as chloramphenicol and mupirocin, which are used in specific cases for the control of staphylococcal infections in pets and companion animals. In addition, plasmids and transposons associated with the respective resistance properties and their ways of spreading between members of the same or different staphylococcal species, but also between staphylococci and other gram-positive bacteria, are described.     

Proposals of the working group "Antibiotic resistance" for the configuration of microtitre plates to be used in routine antimicrobial susceptibility testing of bacterial pathogens from infections of large food-producing animals and mastitis cases

Two layouts for microtitre plates, which should serve for in-vitro susceptibility testing in routine diagnostics, have been set up by the working group "Antibiotic resistance" of the German Society for Veterinary Medicine. One of these layouts was designed for the testing of bacteria from cases of mastitis and the other for bacteria from infections in large food-producing animals. The choice of the antimicrobial agents and their concentrations to be included in these layouts were based on (1) the bacteria frequently associated with the respective diseases/animals, (2) the antimicrobial agents licensed for therapeutic use in these diseases/animals, (3) the currently available breakpoints, and (4) cross-resistances between the antimicrobial agerts so far known to occur in the respective bacteria.     

OIE抗菌药耐药性国际标准

兽用抗菌药耐药性已经成为一个全球普遍关注的公共健康问题,各国际组织都积极采取相应的措施控制耐药性的产生和蔓延。介绍了国际组织世界动物卫生组织OIE制定的五个国际标准,包括协调抗菌药耐药性监督和检测程序指南、畜牧业抗菌药消耗量监测指南、兽用抗菌药慎用指南、抗菌药敏感性检测的实验室方法指南、动物源抗菌药耐药性对公共健康潜在影响的风险分析方法指南,以期为我国政策制定者和决策者参照国际标准制定出符合我国国情的耐药性相关指南。     

Nosocomial infections and antimicrobial resistance in critical care medicine

Objective: To review the human and companion animal veterinary literature on nosocomial infections and antimicrobial drug resistance as they pertain to the critically ill patient. Data sources: Data from human and veterinary sources were reviewed using PubMed and CAB. Human data synthesis: There is a large amount of published data on nosocomially‐acquired bloodstream infections, pneumonia, urinary tract infections and surgical site infections, and strategies to minimize the frequency of these infections, in human medicine. Nosocomial infections caused by multi‐drug‐resistant (MDR) pathogens are a leading cause of increased patient morbidity and mortality, medical treatment costs, and prolonged hospital stay. Epidemiology and risk factor analyses have shown that the major risk factor for the development of antimicrobial resistance in critically ill human patients is heavy antibiotic usage. Veterinary data synthesis: There is a paucity of information on the development of antimicrobial drug resistance and nosocomially‐acquired infections in critically ill small animal veterinary patients. Mechanisms of antimicrobial drug resistance are universal, although the selection effects created by antibiotic usage may be less significant in veterinary patients. Future studies on the development of antimicrobial drug resistance in critically ill animals may benefit from research that has been conducted in humans. Conclusions: Antimicrobial use in critically ill patients selects for antimicrobial drug resistance and MDR nosocomial pathogens. The choice of antimicrobials should be prudent and based on regular surveillance studies and accurate microbiological diagnostics. Antimicrobial drug resistance is becoming an increasing problem in veterinary medicine, particularly in the critical care setting, and institution‐specific strategies should be developed to prevent the emergence of MDR infections. The collation of data from tertiary‐care veterinary hospitals may identify trends in antimicrobial drug resistance patterns in nosocomial pathogens and aid in formulating guidelines for antimicrobial use.     

Antimicrobial drug use and resistance in dogs

Fifteen years (1984-1998) of records from a Veterinary Teaching Hospital were analyzed to determine whether antimicrobial drug resistance in coagulase-positive Staphylococcus spp. (S. aureus, S. intermedius) isolated from clinical infections in dogs has increased, and whether there has been a change in the species of bacteria isolated from urinary tract infections in dogs. In coagulase-positive Staphylococcus spp., a complex pattern showing both increases and decreases of resistance to different classes of antimicrobial drugs was observed, reflecting the changing use of different antimicrobial drug classes in the hospital over a similar period (1990-1999). In canine urinary tract infections identified from 1984 to 1998, an increase in the incidence of multiresistant Enterococcus spp. was apparent, with marginal increases also in incidence in Enterobacter spp. and in Pseudomonas aeruginosa, both of which, like Enterococcus spp., are innately antimicrobial-resistant bacteria. A survey of directors of veterinary teaching hospitals in Canada and the United States identified only 3 hospitals that had any policy on use of "last resort" antimicrobial drugs (amikacin, imipenem, vancomycin). Evidence is briefly reviewed that owners may be at risk when dogs are treated with antimicrobial drugs, as well as evidence that some resistant bacteria may be acquired by dogs as a result of antimicrobial drug use in agriculture. Based in part on gaps in our knowledge, recommendations are made on prudent use of antimicrobial drugs in companion animals, as well as on the need to develop science-based infection control programs in veterinary hospitals.     

Antimicrobial resistance in bacteria from food-producing animals. risk management tools and strategies

The application of antimicrobial agents has proved to be the main risk factor for development, selection and spread of antimicrobial resistance. This link applies to the use of antimicrobial agents in human and in veterinary medicine. Furthermore, antimicrobial-resistant bacteria and resistant genes can be transmitted from animals to humans either by direct contact or via the food chain. In this context, risk management has to be discussed regarding prevention and control of the already existing antimicrobial resistance. One of the primary risk management measures in order to control the development and spread of antimicrobial resistances is by regulating the use of antimicrobial agents and subjecting their use to guidelines. Thereby, the occurrence of antimicrobial resistant bacteria in the human and veterinary habitat can be controlled to a certain degree. There is little information about past attempts to prevent the development of resistances or to control them, and even less is known about the effectiveness or the cost intensiveness of such efforts. Most of the strategies focus on preventing and controlling antimicrobial resistance by means of the reduction or limitation of the use of antimicrobial agents in food-producing animals.     

Towards the establishment and standardization of a veterinary antimicrobial resistance surveillance and monitoring programme in South Africa

The objective of this study was to establish a repeatable, standardized laboratory procedure for monitoring the development of antimicrobial resistance in bacteria isolated from animals and food of animal origin in South Africa, with reagents prepared in-house. The emergence of resistance and the spread of resistant bacteria can be limited by implementing a veterinary antimicrobial drug policy, in which inter alia systematic monitoring and prudent use play essential roles. The bacteria included in this study represented three different categories, namely zoonotic bacteria (Salmonella), indicator bacteria (Escherichia coli, Enterococcus faecalis and Enterococcus faecium) and veterinary pathogens (Mannheimia haemolytica). Thirty isolates of each species were collected with the aim of standardizing the laboratory methodology for a future national veterinary surveillance and monitoring programme. Susceptibility to ten selected antimicrobial drugs was determined by means of minimum inhibitory concentrations (MICs) using the microdilution method. The method according to the National Committee for Clinical Laboratory Standards was used as the standard. Multi-well plates containing varying dilutions of antimicrobial drugs and prepared in-house for MIC determinations, yielded repeatable results. Storage of plates for 2 months at -70 degrees C did not influence results meaningfully. Within this limited sample of bacteria, MIC results did not indicate meaningful resistance against any of the ten selected antimicrobial drugs. The findings of the study will be used to establish a national veterinary antimicrobial resistance surveillance and monitoring programme in South Africa. To allow for international comparison of data, harmonisation of the surveillance and monitoring programme in accordance with global trends is encouraged. Ideally it should be combined with a programme monitoring the quantities of antimicrobial drugs used. The aim is to contribute to slowing down the emergence of resistance and the problems associated with this phenomenon by means of the rational use of antimicrobial drugs.     

26株猪链球菌对抗菌药物的敏感性分析

采用纸片扩散法对兽医临床分离的26株猪链球菌进行了常用抗菌药物的敏感性分析,结果表明,猪链球菌对头孢噻肟及氨苄西林/舒巴坦敏感性最高,敏感菌株均占73.0%;其次是丁胺卡那霉素和盐酸多西环素,敏感菌株分别占57.7%和50.0%;分离菌对四环素、庆大霉素、磺胺二甲氧嘧啶表现出较强的耐药性,耐药率分别为92.3%、69.2%、69.2%;此外,分离菌对氯霉素、头孢他啶、克林霉素、磺胺嘧啶钠、复方新诺明也表现出一定的耐药性,耐药率均为57.7%。分离的猪链球菌对部分兽医临床常用抗菌药物产生了较为严重的耐药性,应引起重视。     

Antimicrobial resistance in swine production

Large amounts of antimicrobial agents are still being used in modern swine production in many countries around the world. This facilitates the emergence and development of antimicrobial resistance. Bacteria causing infections in swine have in several cases acquired resistance to a number of the agents most commonly used for treatment, making it difficult to predict the efficacy of different antimicrobial agents without prior susceptibility testing. This review gives an overview of recent susceptibility data from different parts of the world and discusses the importance of the development of resistance not only in the treatment of infections in swine but also taking into account the human health implications of antimicrobial resistance.     

鸡白痢沙门氏菌耐药性的监测研究

采用WHO推荐的Kirby-Bauer法对我国部分地区1962－1999年间346株鸡白痢沙门氏菌进行了药物敏感性测定。结果表明，在近40年时间里，鸡白痢沙门氏菌对氨苄青霉素、壮观霉素、复方磺胺、磺胺异恶性唑、甲氧苄胺嘧啶、羧苄青霉素、四环素、链霉素、青霉素的耐药率显著增强（P＜0.01)。菌株的多重耐药率明显增加。60年代以二耐菌株为主（37.0%)，70年代以四耐、五耐菌株居多（60.5%)，80年代五耐、六耐、七耐菌株占大多数（80.2%)，90年代仅七耐以上菌株就达83.7%。不同年代菌株显示出不尽相同的耐药谱，且呈现出不断增宽的趋势。通过流行病学调查发现，60年代至90年代，鸡白痢沙门氏菌对青霉素、链霉素、磺胺类药物、四环素、卡那霉素、氯霉素、庆大霉素等药物的耐药性大多呈现出显著增强的变化趋势，这充分说明细菌耐药性的形成和发展与抗菌药物长期反复使用有着极为密切的关系。研究对于指导临诊合理用药及控制疾病流行具有重要的现实意义。     

Antimicrobial resistance.

Development of antimicrobial resistance is an inevitable consequence of exposure of microorganisms to antimicrobial agents. Although emergence of resistance cannot be prevented, it can be retarded by minimizing use of antimicrobial agents and avoiding selection of relatively resistant pathogenic and nonpathogenic strains caused by exposure to tissue concentrations that confer a competitive advantage. Most attention in veterinary medicine has focused on the emergence of resistance in food-borne pathogens, with relatively little attention being devoted to small companion animals, despite the frequent use of antimicrobial agents in these animals, evidence that resistance is emerging, and potential for transfer of resistance between companion animals and people. To retard further emergence of resistance in small companion animals, it is imperative that surveillance programs be instituted to monitor development of resistance.     

9种抗菌药物对大肠埃希菌最小抑菌浓度的测定

畜禽养殖中会用到一些抗菌药物,而药物选用需要进行药物敏感性试验,目前市售的药敏纸片多为人用抗菌药物,为了制备兽用抗菌药物的药敏纸片,需要对药物的最小抑菌浓度(MIC)进行测定。该研究筛选了国家兽用药物名录中规定的、兽医临床上常用6类共9种抗菌药物(头孢噻呋钠、硫酸链霉素、强力霉素、土霉素、氨苄西林、氟苯尼考、替米考星、硫酸新霉素、青霉素),以大肠埃希菌为指标菌,通过微量肉汤稀释法分别测定了9种常用抗菌药物的最小抑菌浓度(MIC)。结果表明,头孢噻呋钠、硫酸链霉素、强力霉素、土霉素、氨苄西林、氟苯尼考、替米考星、硫酸新霉素、青霉素对指标菌的MIC值分别为为2、4、4、16、8、16、64、32、250μg/mL。研究结果为这些药物的临床应用和药敏纸片的研制提供了基础资料。     

Antimicrobial susceptibility pattern of Helicobacter suis strains

Helicobacter suis is a very fastidious porcine gastric pathogen, which is also considered to be of zoonotic importance. In vitro antimicrobial susceptibility cannot be determined using standard assays, as this agent only grows in a biphasic medium with an acidic pH. Therefore, a combined agar and broth dilution method was used to analyse the activity of nine antimicrobial agents against nine H. suis isolates. After 48 h microaerobic incubation, minimal inhibitory concentrations (MICs) were determined by software-assisted calculation of bacterial growth. Only for enrofloxacin a bimodal distribution of MICs was demonstrated, indicating acquired resistance in one strain, which showed an AGT→AGG (Ser→Arg) substitution at codon 99 of gyrA. In conclusion, the assay developed here is suitable for determination of the antimicrobial susceptibility of H. suis isolates, although activity of acid sensitive antimicrobial agents may be higher than predicted from MIC endpoints.     

Antimicrobial susceptibility testing of bacteria isolated from animals: methods for in-vitro susceptibility testing and their suitability with regard to the generation of the most useful data for therapeutic applications

In-vitro susceptibility testing provides valuable informations for choosing the most suitable antimicrobial agent for the control of bacterial infections in animals. Different diffusion and dilution methods, as conducted according to various approved performance standards, can be used to determine the in-vitro susceptibility of bacterial pathogens. In the present article, problems are discussed which arise from the use of different methods and the difficulty to interpret such results. While most approved performance standards were designed for testing of bacteria from human sources, the NCCLS document M31-A2 exclusively focusses on susceptibility testing of bacteria isolated from animals and--in contrast to all other standards--includes veterinary specific breakpoints for a number of antimicrobial agents used in veterinary medicine. Therefore, performance of in-vitro susceptibility testing of veterinary pathogens should follow the recommendations given in the NCCLS document M31-A2. The microdilution method is recommended as the method of choice for susceptibility testing. The result of a microdilution test is given as the minimum inhibitory concentration (MIC). This value provides a quantitative result which precisely indicates the degree of susceptibility of the tested bacterial strain and in return gives the veterinarian a clear guidance whether therapeutic intervention with the antibiotic in question will be successful.     

内蒙古地区羊源大肠杆菌耐药性研究

为确定内蒙古地区羊源大肠杆菌的耐药表型及其耐药基因的流行情况,本研究采用微量稀释法测定了内蒙古地区108株羊源大肠杆菌对13种临床常用抗菌药物的最小抑菌浓度。结果显示,分离菌株对阿莫西林、头孢噻吩、磺胺甲唑、黏菌素的耐药率最高,均达100.0%,对阿莫西林/克拉维酸、四环素、环丙沙星的耐药率在50%~80%之间,对头孢噻肟、美洛培南、新霉素的耐药率均低于10%,较为敏感。108株羊源大肠杆菌中耐7种以上药物的菌株占94.4%,其中15.6%菌株对13种抗菌药物耐药,只有1株菌对所有抗菌药物敏感。采用PCR方法对羊源大肠杆菌分离株所携带的6种相关耐药基因进行检测,结果显示,6种耐药基因中的4种耐药基因bla_TEM、proP-2、sul-Ⅰ、ampG检出率超过50%,耐药基因aph（3'）-Ⅰ携带率达40%,只有耐药基因aac（3）-Ⅱ检出率仅为5.5%。由此可见,内蒙古地区羊源大肠杆菌对13种抗菌药物产生了不同程度的耐药性,且存在严重的多重耐药情况,羊源大肠杆菌分离株携带aph（3'）-Ⅰ、sul-Ⅰ、ampG、bla_TEM、proP-2、aac（3）-Ⅱ耐药基因。     

Development of antimicrobial drug resistance in rectal Escherichia coli isolates from dogs hospitalized in an intensive care unit

OBJECTIVE: To determine whether duration of hospitalization in the intensive care unit (ICU) of a veterinary teaching hospital was associated with prevalence of antimicrobial resistance among rectal Escherichia coli isolates from dogs, whether antimicrobial treatment was associated with prevalence of antimicrobial resistance, and whether there were associations among antimicrobial drugs to which isolates were resistant. DESIGN: Prospective observational study. ANIMALS: 116 dogs hospitalized in an ICU for >or= 3 days. PROCEDURES: Rectal swab specimens were obtained every 3 days and submitted for bacterial culture for E coli. Isolates were tested for susceptibility to 12 antimicrobial agents by means of disk diffusion. RESULTS: For each additional day that a dog was hospitalized in the ICU, the odds of being colonized with an E coli isolate resistant to 1 or more of the 12 antimicrobials tested increased by a factor of 1.5, independent of antimicrobial treatment. Dogs that were treated with enrofloxacin were 25.6 times as likely to be colonized by a quinolone-resistant E coli strain as were dogs that did not receive any antimicrobials. Significant correlations were found for resistance to agents in the extended-spectrum cephalosporin group and the quinolone group. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that the proportion of rectal E coli isolates obtained from dogs housed for >or= 3 days in a veterinary teaching hospital ICU that were resistant to antimicrobial agents increased as the duration of hospitalization in the ICU increased. Thus, ICU hospitalization time should be as short as possible to prevent development of antimicrobial resistance among rectal E coli isolates.     

Study on the Drug-resistance of Escherichia coli Isolated from Sheep in Inner Mongolia

In order to determine the drug resistance phenotype and prevalence of drug resistance genes in Escherichia coli (E.coli) isolated from sheep in Inner Mongolia, the minimum inhibitory concentrations (MICs) of the isolates to antibiotics commonly used in veterinary clinical were detected by micro-dilution method in vitro. The results showed that the highest resistance rates of the isolates to amoxicillin,cephalothin,sulfamethoxazole and polymyxin were up to 100.0%,respectively.To amoxicillin-clavulanic, tetracycline and ciprofloxacin were 50% to 80%. These isolates were sensitive to cefotaxime, meropenem trihydrate, neomycin, and their resistance rates were all less than 10%. Among the 108 strains of E.coli from sheep, 94.4% of them were resistant to more than 7 antimicrobial agents,15.6% of them were resistant to 13 antimicrobial agents, only one strain was sensitive to all antimicrobial agents. Six kinds of resistance genes among the 108 E.coli isolates were detected by PCR method.The results showed that detection rates of 4 kinds of drug resistance genes including bla_TEM, proP-2, sul-Ⅰ and ampG were all over 50%,the detection rate of resistance gene aph (3')-Ⅰ was up to 40%, only resistance gene aac(3)-Ⅱ detection rate was 5.5%. Thus, the sheep E.coli isolates in Inner Mongolia produced various degrees of resistance to 13 kinds of antibiotics, and their multi-drug resistances were very serious. They carried aph(3')-Ⅰ, sul-Ⅰ, ampG, bla_TEM, proP-2 and aac(3)-Ⅱ resistance genes.     

beta-Lactam resistance and beta-lactamases in bacteria of animal origin

beta-Lactams are among the most clinically important antimicrobials in both human and veterinary medicine. Bacterial resistance to beta-lactams has been increasingly observed in bacteria, including those of animal origin. The mechanisms of beta-lactam resistance include inaccessibility of the drugs to their target, target alterations and/or inactivation of the drugs by beta-lactamases. The latter contributes predominantly to beta-lactam resistance in Gram-negative bacteria. A variety of beta-lactamases have been identified in bacteria derived from food-producing and companion animals and may further serve as a reservoir for beta-lactamase-producing bacteria in humans. While this review mainly describes beta-lactamases from animal-derived Escherichia coli and Salmonella spp., beta-lactamases from animal-derived Campylobacter spp., Enterococcus spp., Staphylococcus spp. and other pathogens are also discussed. Of particular concern are the increasingly-isolated plasmid-encoded AmpC-type CMY and extended-spectrum CTX-M beta-lactamases, which mediate acquired resistance to extended-spectrum beta-lactams. The genes encoding these enzymes often coexist with other antimicrobial resistance determinants and can also be associated with transposons/integrons, increasing the potential enrichment of multidrug resistant bacteria by multiple antimicrobial agents as well as dissemination of the resistance determinants among bacterial species. Characterization of beta-lactam-resistant animal-derived bacteria warrants further investigation of the type and distribution of beta-lactamases in bacteria of animal origin and their potential impact on human medicine.     

Antimicrobial susceptibility of Pseudomonas aeruginosa from dogs and cats as well as Arcanobacterium pyogenes from cattle and swine as determined in the BfT-GermVet monitoring program 2004-2006

During the BfT-GermVet monitoring program, Pseudomonas (P) aeruginosa from dogs and cats (n = 99) as well as Arcanobacterium (A.) pyogenes from cattle and swine (n = 90) were examined for their antimicrobial susceptibility. In general, P. aeruginosa is known to be resistant against many antimicrobial agents whereas A. pyogenes is thought to be susceptible to most agents in-vitro. However, representative and actual minimum inhibitory concentration (MIC) values are missing for both veterinary pathogens. In the present study, MIC values were determined and categorized according to the recommendations given in the Clinical and Laboratory Standards Institute (CLSI) documents M31-A2 and M31-S1. For susceptibility testing of A. pyogenes, the CLSI methodology was slightly modified. Specific breakpoints were not available for most of the antimicrobial agents tested. P. aeruginosa isolates from infections of the skin, ear and mouth as well as the urinary and genital tract of dogs and cats were either resistant or exhibited high MIC values to most antimicrobial agents tested. However, gentamicin resistant isolates were observed in only 27% and 11% (intermediate isolates 29% and 39%), respectively. For the same bacterium/host animal/organ system combinations, enrofloxacin resistance was detected in only 24% and 11% of the isolates (intermediate isolates 49% and 61%). For A. pyogenes, resistance was most prevalent against tetracycline (33%-56%, bovine and porcine isolates) and sulfonamides (26%-40%, bovine isolates).