2022年北京市宠物源细菌耐药性监测浅析

为了解北京市宠物源细菌的抗菌药物耐药情况,2022年,笔者对北京市四个城区的四家宠物医院的犬、猫共计50份样本的肛拭子进行了研究。试验对样品中的大肠杆菌和肠球菌首先进行了分离培养和质谱鉴定,然后采用微量肉汤稀释法分析分离菌株的耐药表型。结果共分离出大肠杆菌25株、肠球菌25株（屎肠球菌14株、粪肠球菌11株）。大肠杆菌耐药率最高的2种抗菌药为四环素和氨苄西林,多重耐药菌占44%;肠球菌耐药情况较严重,粪肠球菌耐药率最高的抗菌药物为磺胺异噁唑,屎肠球菌耐药率最高的抗菌药物为磺胺异噁唑、头孢西丁和红霉素,二者多重耐药菌占分离株总数的100%。综上,北京地区宠物源大肠杆菌、肠球菌的耐药情况较为严峻,且多重耐药现象突出,需要加强对宠物抗菌药使用的监督与管理。     

Antimicrobial resistance in Swiss laying hens, prevalence and risk factors

Antimicrobial resistance is an emerging concern to public health, and food-producing animals are known to be a potential source for transmission of resistant bacteria to humans. As legislation of the European Union requires to ban conventional cages for the housing of laying hens on the one hand, and a high food safety standard for eggs on the other hand, further investigations about the occurrence of antimicrobial resistance in alternative housing types are required. In this study, we determined antimicrobial resistance in indicator bacteria from 396 cloacal swabs from 99 Swiss laying hen farms among four alternative housing types during a cross-sectional study. On each farm, four hens were sampled and exposure to potential risk factors was identified with a questionnaire. The minimal inhibitory concentration was determined using broth microdilution in Escherichia coli (n=371) for 18 antimicrobials and in Enterococcus faecalis (n=138) and Enterococcus faecium (n=153) for 16 antimicrobials. All antimicrobial classes recommended by the European Food Safety Authority for E. coli and enterococci were included in the resistance profile. Sixty per cent of the E. coli isolates were susceptible to all of the considered antimicrobials and 30% were resistant to at least two antimicrobials. In E. faecalis, 33% of the strains were susceptible to all tested antimicrobials and 40% were resistant to two or more antimicrobials, whereas in E. faecium these figures were 14% and 39% respectively. Risk factor analyses were carried out for bacteria species and antimicrobials with a prevalence of resistance between 15% and 85%. In these analyses, none of the considered housing and management factors showed a consistent association with the prevalence of resistance for more than two combinations of bacteria and antimicrobial. Therefore we conclude that the impact of the considered housing and management practices on the egg producing farms on resistance in laying hens is low.     

我国18个省份猪源肠球菌耐药调查及分析

为研究我国猪源肠球菌的耐药情况,本研究采集18个省市猪养殖场的粪便样品836份进行了肠球菌的分离鉴定,并通过琼脂稀释法测定11种抗菌药物的最小抑菌浓度。结果显示:从836份样品中共分离出225株肠球菌,其中屎肠球菌106株,粪肠球菌56株,小肠肠球菌34株,鹑鸡肠球菌17株,铅黄肠球菌7株,坚忍肠球菌4株,Enterococcus thailandicus 1株。所有的肠球菌对被检抗菌药物耐药程度由高到低依次是四环素(98.2%)、阿米卡星(94.7%)、多西环素(89.3%)、米诺环素(88.9%)、氟苯尼考(88.4%)、红霉素(86.2%)、环丙沙星(63.1%)、氨苄西林(33.8%)、氯霉素(29.8%),未检测到万古霉素和利奈唑胺耐药菌株。除氨苄西林、万古霉素、利奈唑胺外,粪肠球菌对其余8种被检抗菌药物耐药率均高于屎肠球菌。小肠肠球菌对阿米卡星、红霉素、四环素、多西环素、米诺环素5种抗菌药物的耐药率高于屎肠球菌。肠球菌的多重耐药情况集中在6耐、7耐和8耐,占肠球菌总数的72.9%。本研究表明我国猪源肠球菌对常见抗菌药物耐药率较高,应引起更多关注。     

The effect of rehabilitation of northern elephant seals (Mirounga angustirostris) on antimicrobial resistance of commensal Escherichia coli

The aim of this study was to determine if antimicrobial drug use increases resistance of commensal gastrointensinal Escherichia coli of wild northern elephant seals (Mirounga angustirostris) treated in rehabilitation, and, if so, identify the risk factors involved. Minimum inhibitory concentration (MIC) levels of twelve antimicrobial drugs were determined for 289 E. coli isolates from 99 seals sampled at admission and 277 isolates obtained at release from rehabilitation using broth microdilution. Prevalence of E. coli antimicrobial resistance, MIC(50), MIC(90), and clustering of MIC values were determined for seals and the data were analyzed using Fisher's exact test, ordinal logistic regression and negative binomial regression. At release from rehabilitation 77.8% of the seals had antimicrobial resistant E. coli compared to 38.4% of the seals at admission. The MIC(90) for amoxicillin-clavulanic acid, chloramphenicol, enrofloxacin, ticarcillin-clavulanic acid, and trimethoprim-saulfamethoxazole were at levels considered to be sensitive at admission but they increased to levels of resistance at release. E. coli were grouped into four clusters by their MIC values, with increasing levels of resistance going from Cluster 1 to 4. A primary risk factor associated with the probability of a seal having E. coli in Clusters 3 and 4 was time in rehabilitation, regardless of whether the animal received treatment with antimicrobial drugs, suggesting nosocomial infection. The results of this study provide evidence that increased levels of hygiene and appropriate use of antimicrobial therapy might be important in the rehabilitation of wild animals to prevent rise in the prevalence of antimicrobial resistant bacteria.     

Antimicrobial and heavy metal resistance in commensal enterococci isolated from pigs

Antibiotic resistance in animal isolates of enterococci is of public health concern because of the risk of transfer of antibiotic resistance isolates or resistance determinants to consumers via the food chain. In this study, phenotypic and genotypic resistance in 192 pig isolates of enterococci to ampicillin, avilamycin, avoparcin, bacitracin, flavophospholipol, gentamicin, narasin, tetracycline, tiamulin, tylosin, vancomycin, virginiamycin, copper and zinc were investigated by susceptibility test and molecular methods. Resistance rates varied between the species but all isolates were susceptible to ampicillin, avilamycin, avoparcin, gentamicin and narasin but resistant to tetracycline and tylosin and intermediately resistant to copper. Only Enterococcus gallinarum and Enterococcus casseliflavus were resistant to vancomycin and virginiamycin resistance was present in less than half the Enterococcus faecium isolates. Zinc resistance was largely confined to Enterococcus faecalis but bacitracin resistance was uncommon in E. faecalis in comparison with the other species. Tiamulin resistance was common in all species except E. casseliflavus. Resistance to flavophospholipol was detected in most E. faecium isolates and in a high proportion of E. gallinarum, E. casseliflavus and E. hirae/durans but was only found in one isolate of E. faecalis. No tetO, rplC, rplD, vanA, vanB, vatA and vatD genes were found. The presence of ermB, tetL, tetM, tcrB, aac6-aph2, tetK, tetS, vanC1, vanC2, lsaA, lsaB and vatE varied between the species and largely corresponded to the susceptibility phenotype. The findings show that resistance to antibiotics of high clinical significance for nosocomial Enterococcus infections is absent, whereas antimicrobial resistance was detected for some other antibiotics including bacitracin, flavophospholipol, tetracycline, tiamulin, tylosin and virginiamycin.     

广东地区猪源大肠杆菌耐药性及其系统发育群的研究简

为分析广东地区猪源大肠杆菌耐药性及其系统进化背景,试验于2011年从患病猪分离出264株大肠杆菌,包括粪便源131株和肝源133株,采用琼脂稀释法测定18种抗菌药物的敏感性,多重PCR法确定系统进化关系,并对不同来源菌株的耐药性和系统进化背景进行分析比较。结果发现,大肠杆菌多重耐药现象严重,共存在128种耐药谱型。分离菌株对氨苄西林、复方新诺明、萘啶酸和四环素高度耐药,对黏菌素、头孢他啶和阿米卡星较敏感。种群进化分析结果表明,分离菌株主要分布在共生型的A组和B1组（91.66%）,且不同来源菌株的种群进化分布差异不显著(P＞0.05)。结合药敏试验结果发现,不同进化组别的分离菌株对特定抗菌药物的耐药性存在显著差异（P＜0.05）。通过对大肠杆菌耐药性及其系统发育群进行综合分析,为兽医临床用药提供依据。     

Phenotypic and genotypic characterization of antimicrobial resistance in faecal enterococci from wild boars (Sus scrofa)

The objective was to study the prevalence of antimicrobial resistance and the mechanisms implicated in faecal enterococci of wild boars in Portugal. One hundred and thirty-four enterococci (67 E. faecium, 54 E. hirae, 2 E. faecalis, 2 E. durans and 9 Enterococcus spp.) were recovered from 67 wild boars (two isolates/sample), and were further analysed. High percentages of resistance were detected for erythromycin, tetracycline, and ciprofloxacin (48.5%, 44.8%, and 17.9%, respectively), and lower values were observed for high-level-kanamycin, -streptomycin, chloramphenicol, and ampicillin resistance (9%, 6.7%, 4.5%, and 3.7%, respectively). No isolates showed vancomycin or high-level-gentamicin resistance. The erm(B), tet(M), aph(3')-IIIa, and ant(6)-I genes were demonstrated in all erythromycin-, tetracycline-, kanamycin-, and streptomycin-resistant isolates, respectively. Specific genes of Tn916/Tn1545 and Tn5397 transposons were detected in 78% and 47% of our tet(M)-positive enterococci, respectively. The tet(S) and tet(K) genes were detected in one isolate of E. faecium and E. hirae, respectively. Three E. faecium isolates showed quinupristin-dalfopristin resistance and the vat(E) gene was found in all of them showing the erm(B)-vat(E) linkage. Four E. faecium isolates showed ampicillin-resistance and all of them presented seven amino acid substitutions in PBP5 protein (461Q-->K, 470H-->Q, 485M-->A, 496N-->K, 499A-->T, 525E-->D, and 629E-->V), in relation with the reference one; a serine insertion at 466' position was found in three of the isolates. Faecal enterococci from wild boars harbour a variety of antimicrobial resistance mechanisms and could be a reservoir of antimicrobial resistance genes and resistant bacteria that could eventually be transmitted to other animals or even to humans.     

Monitoring of antimicrobial resistance in healthy dogs: first report of canine ampicillin-resistant Enterococcus faecium clonal complex 17

National surveillance programs on antimicrobial usage and antimicrobial resistance in animals have been established in various countries but few of them include bacteria from pets. The objectives of this study were to assess the prevalence of antimicrobial resistance in healthy dogs and to search for resistance phenotypes of clinical relevance. Escherichia coli and Enterococcus spp. were isolated from faecal swabs obtained from 127 dogs. Disk diffusion was used to measure antimicrobial susceptibility in 117 Escherichia coli, 10 Enterococcus faecium and 51 Enterococcus faecalis of canine origin. Resistance was relatively low compared with food animal species in Denmark. All Escherichia coli isolates were susceptible to broad-spectrum aminopenicillins, third generation cephalosporins and fluoroquinolones. Despite the low prevalence of resistance, statistical analysis of questionnaire data revealed a significant association (p=0.02) between recent antimicrobial treatment and resistance in Escherichia coli. Interestingly, two dogs were found to shed Enterococcus faecium resistant to ampicillin. Multilocus sequence typing of these isolates indicated that the two isolates belonged to sequence types associated with human nosocomial infections, and one (ST-192) was genetically related to human epidemic clonal complex 17. The detection of ampicillin-resistant Enterococcus faecium warrants further studies on the prevalence of these bacteria in dogs and on the possible implications to both animal and human health. The results suggest that distinct methods for detection and assessment of antimicrobial resistance in animals should be considered depending on the target animal species and the purposes of the study.     

Prevalence of and resistance to anti-microbial drugs in selected microbial species isolated from bulk milk samples

The prevalence of strains of Staphylococcus aureus, coagulase-negative (CN) staphylococci, Listeria monocytogenes, Escherichia coli, Enterococcus faecalis, E. faecium and Bacillus cereus, was investigated in 111 bulk milk samples. Staphylococcus aureus was isolated from 38 samples, CN staphylococci from 63 samples, E. coli from 49 samples, E. faecalis or E. faecium from 107 samples, and L. monocytogenes from two samples. Bacillus cereus was not found in any of the samples and three samples were free of any of the selected species. Sensitivity to the anti-microbial drugs amikacin, ampicillin, ampicillin + sulbactam, cephalothin (CLT), cephotaxime, clindamycin, chloramphenicol (CMP), co-trimoxazole, erythromycin (ERY), gentamicin, neomycin, norfloxacin, oxacillin, penicillin, streptomycin (STR), tetracycline (TTC) and vancomycin was tested using the standard dilution technique. Minimum inhibitory concentration (MIC) characteristics (MIC50, MIC90, MIC range) were determined for each microbial species. Resistance against one or more anti-microbial drugs was found in 93% of S. aureus, 40% of CN staphylococci, 73% of E. coli, 88% of E.faecalis, 55% of E.faecium, and one L. monocytogenes strain. Most of the strains, particularly enterococci, were resistant to STR, TTC, and ERY (MIC50 4 microg/ml). A high percentage of staphylococci were resistant to beta-lactam antibiotics. High resistance to CLT was found in 11 strains of E. coli (MIC 256 microg/ml) and strains resistant to CMP (MIC90 16 microg/ml) were detected. The highest numbers of resistance phenotypes were found in E. coil (16) and CN staphylococci (12). Eighteen identical resistance phenotypes were demonstrated in indicator bacteria (E. coli, E. faecalis, E. faecium) and pathogens (S. aureus, CN staphylococci) isolated from the same bulk milk sample. The obtained resistance data were matched against the herd owners' information on therapeutic use of the drugs. This confrontation could not explain the findings of strains resistant to ERY or CMP. Our findings are evidence of selection of resistant strains among not only pathogenic agents, but also among indicator bacteria which can become significant carriers of transmissible resistance genes.     

Susceptibility of Escherichia coli and Enterococcus faecium isolated from pigs and broiler chickens to tetracycline degradation products and distribution of tetracycline resistance determinants in E. coli from food animals

One hundred Escherichia coli isolates from diseased and healthy pigs, cattle and broiler chickens were screened for the presence of tetracycline resistance genes tet(A), (B), (C), (D) or (E). The tet(A) gene was the most abundant (71% of the 100 isolates) followed by tet(B) (25%). The predominance of tet(A) and tet(B) applied to all three animal species, and there was no difference between the distribution of tet(A) and tet(B) genes among non-pathogenic and pathogenic E. coli in any of the animal species. The susceptibility of 20 of these isolates together with 10 tetracycline sensitive E. coli and 18 tetracycline resistant and 10 sensitive Enterococcus faecium to tetracyclines and tetracycline degradation products was determined. The resistant isolates showed reduced resistance to anhydrotetracycline, 4-epi-anhydrotetracycline, anhydrochlortetracycline and 4-epi-anhydrochlortetracycline. In general both the tetracycline resistant and susceptible E. faecium were more susceptible to the compounds tested than E. coli.