食源性沙门氏菌耐药性检测及相关质粒

[目的]测定390株沙门氏菌的抗生素药敏性,研究部分多重耐药株中质粒与其宿主耐药表型之间的关系及其在接合过程中对耐药性水平转移的影响.[方法]使用选择性培养基分离到沙门氏菌并通过PCR确认后,按照琼脂稀释法测定分离株对供试抗生素的药敏性,试剂盒提取代表性多重耐药株中的质粒,HindⅢ酶切,DPS软件分析电泳后质粒图谱.通过接合试验研究质粒在抗生素抗性水平转移中的作用.[结果]沙门氏菌分离株对四环素耐药最为普遍(58.2%),其次为链霉素(42.8%)、卡那霉素(39%)和氨苄青霉素(38.2%),对头孢西丁、氯霉素、庆大霉素、头孢曲松、阿莫西林甲氧苄啶、头孢替呋钠和萘啶酮酸的耐药率分别为27.2%、26.9%、21%、19%、18.2%、17.9%、14.6%和12.3%.抗性质粒编码的相同或相似沙门氏菌耐药表型与其中所含的耐药质粒并不呈现出严格的对应关系.质粒携带的抗性基因可通过接合作用转移,接合效率在2.4×10-4到5.6×10-1之间.[结论]食源性沙门氏菌对常用抗生素的多重耐药已经成为普遍现象,抗性质粒的同源性与其宿主耐药表型无直接相关性,其携带的耐药基因可通过接合作用在不同细菌种属之间高频传递.     

Salmonella serotypes isolated from pet chews in New Zealand

AIMS: To survey the prevalence of Salmonella in imported and domestic pet chews for assessing their potential in introducing novel pathogenic and antimicrobial resistant Salmonella serotype clones into New Zealand, and as vehicles of salmonellosis in the domestic home environment. METHODS AND RESULTS: Three hundred samples, each of imported and domestic pet chews, were examined bacteriologically for the presence of Salmonella. Salmonella cells in the pre-enrichment culture were concentrated by using Dynabeads, and then selective enrichment and plating were performed by a method described in the Bacteriological and Analytical Manual, USFDA. Salmonella was isolated from 16 (5.3%) of the imported and 20 (6.7%) of the domestic pet chews, but the prevalences of Salmonella in imported and domestic products were not significantly different. All Salmonella isolates were serotyped and genotyped by pulsed-field gel electrophoresis and antimicrobial susceptibility determined by the Clinical and Laboratory Standards Institute disc diffusion methods. Salmonella Borreze has never been recorded earlier in New Zealand and was detected from Australian raw hide. Three isolates of Salmonella London were resistant to ampicillin and gentamicin, and two isolates of Salmonella Infantis were resistant to nalidixic acid, one of which was also resistant to streptomycin. CONCLUSIONS: Novel pathogenic and antimicrobial-resistant Salmonella are being introduced into New Zealand through the import of pet chews. This indicates that pet chews are a potential source of exposure to Salmonella in the domestic home environment. SIGNIFICANCE AND IMPACT OF THE STUDY: Contaminated pet chews are potential sources of Salmonella infection for domestic pets, and humans are at risk of exposure either directly by contact through handling or inadvertently by cross-contamination of food or food-contact surfaces in home environments.     

Antibiotic resistant Escherichia coli and Salmonella in Russian rooks (Corvus frugilegus) wintering in the Czech Republic

AIMS: To characterize antibiotic resistant Escherichia coli and Salmonella isolates in rooks wintering in the Czech Republic. METHODS AND RESULTS: Three hundred and sixty-three faeces samples from rooks were examined for antibiotic resistant Escherichia coli and Salmonella. Altogether 13.7%E. coli isolates were resistant to antimicrobial agents tested. The dominant type of resistance was to tetracycline. Resistant E. coli isolates were examined for antibiotic resistance genes and class 1 integrons. Five of 29 antibiotic resistant isolates possessed the int1 gene. Nine Salmonella isolates (2.5%) were found in rook faeces. All the isolates belonged to serotype Salmonella enterica serovar Enteritidis phage type PT8 and PT23. CONCLUSIONS: The study suggests that rooks can be infected by antibiotic resistant E. coli and Salmonella isolates, probably reflecting the presence of such isolates in their sources of food and/or water in the environment. SIGNIFICANCE AND IMPACT OF THE STUDY: Rooks can serve as reservoirs and vectors of antibiotic resistant E. coli and Salmonella isolates and potentially transmit these isolates over long distances.     

Effect of previous handling experiences on responses of dairy calves to routine husbandry procedures

The nature of human–animal interactions is an important factor contributing to animal welfare and productivity. Reducing stress during routine husbandry procedures is likely to improve animal welfare. We examined how the type of early handling of calves affected responses to two common husbandry procedures, ear-tagging and disbudding. Forty Holstein–Friesian calves (n = 20/treatment) were exposed to one of two handling treatments daily from 1 to 5 weeks of age: (1) positive (n = 20), involving gentle handling (soft voices, slow movements, patting), and (2) negative (n = 20), involving rough handling (rough voices, rapid movements, pushing). Heart rate (HR), respiration rate (RR) and behaviour (activity, tail flicking) were measured before and after ear-tagging and disbudding (2 days apart). Cortisol was measured at −20 (baseline), 20 and 40 min relative to disbudding time. There were no significant treatment differences in HR, RR or behaviour in response to either procedure. However, the following changes occurred across both treatment groups. HR increased after disbudding (by 14.7 ± 4.0 and 18.6 ± 3.8 bpm, positive and negative, respectively; mean ± s.e.m.) and ear-tagging (by 8.7 ± 3.1 and 10.3 ± 3.0 bpm, positive and negative, respectively). After disbudding, there was an increase in RR (by 8.2 ± 3.4 and 9.3 ± 3.4 breaths/min, positive and negative, respectively), overall activity (by 9.4 ± 1.2 and 9.9 ± 1.3 frequency/min, positive and negative, respectively) and tail flicking (by 13.2 ± 2.8 and 11.2 ± 3.0 frequency/min, positive and negative, respectively), and cortisol increased from baseline at 20 min post procedure (by 10.3 ± 1.1 and 12.3 ± 1.1 nmol/l positive and negative, respectively). Although we recorded significant changes in calf responses during ear-tagging and disbudding, the type of prior handling had no effect on responses. The effects of handling may have been overridden by the degree of pain and/or stress associated with the procedures. Further research is warranted to understand the welfare impact and interaction between previous handling and responses to husbandry procedures.     

Prevalence of the multiple antibiotic resistance operon (marRAB) in the genus Salmonella

The multiple antibiotic resistance operon (marRAB) is a member of the multidrug resistance (mdr) systems. Similar to other mdr systems, this operon when induced encodes resistance to structurally and functionally unrelated antibiotics. marRAB has been shown to be conserved in the family Enterobacteriaceae, but within the genus Salmonella certain species appeared to be lacking this operon. To investigate how conserved the marRAB operon was in Salmonella, 30 veterinary isolates were examined by PCR, Southern blot, and dot blot analysis. Using DNA primers based on the marRAB operon of S. typhimurium, a predicted 2.3-kb amplicon resulted after PCR in 16 of the 30 organisms. The 2.3-kb DNA band from S. enteritidis was cloned and sequenced and shown to possess 99% sequence homology to marRAB from S. typhimurium. Using a labeled marRAB gene probe from S. enteritidis, Southern blot and dot blot analysis confirmed the presence of the operon in all 30 Salmonella species examined. Furthermore, when these isolates were induced with low levels of either tetracycline or chloramphenicol, increased antimicrobial resistance was observed to structurally and functionally unrelated antibiotics. Thus, the widespread occurrence of the marRAB locus in this genus prescribes judicious use of antimicrobials to avoid induction of a mdr phenotype.     

Effects of isovalerate supplements on morphology and functional gene expression of rumen mucosa in pre- and post-weaning dairy calves

Isovalerate supplements could stimulate rumen development by improving morphology and function of rumen mucosa, and then promote the growth of calves. This study was done to evaluate the effects of isovalerate supplements on morphology and functional gene expression of rumen mucosa in dairy calves. In total, 48 Chinese Holstein male calves with 15 days of age and 45.1±0.36 kg of BW were randomly assigned to four groups. The treatments were: control, low-isovalerate, moderate-isovalerate and high-isovalerate with 0, 3, 6 and 9 g isovalerate per calf per day, respectively. Supplementary isovalerate was hand-mixed into milk in pre-weaning calves and into concentrate portion in post-weaning calves. The study consisted of a 15-day-adaptation period and a 60-day-sampling period. Calves were weaned at 60 days of age. Three calves were slaughtered from each of the four treatments at 30, 60 and 90 days of age. The weight of body and stomach were measured, samples of ruminal tissues and blood were analyzed. Total stomach weight, total stomach to BW ratio, rumen wall and keratinized layer thickness, serum growth hormone and IGF-1 for both pre- and post-weaning calves increased linearly with increasing isovalerate supplements. Rumen to total stomach weight ratio, the length and width of rumen papillae, and serum β-hydroxybutyrate increased linearly for post-weaning calves. However, abomasum weight to total stomach weight ratio decreased linearly for both pre- and post-weaning calves. The relative messenger RNA expression for growth hormone receptor, IGF-1 receptor and 3-hydroxy-3-methylglutaryl-CoA synthase 1 in rumen mucosa increased linearly for post-weaning calves. Our results suggested that isovalerate supplements promoted rumen development in a dose-dependent manner. The optimum dose was 6.0 g isovalerate per calf per day.     

鸡蛋生产链中沙门氏菌对抗生素及消毒剂的耐药性研究

为研究鸡蛋生产链中沙门氏菌的污染情况及抗生素、消毒剂耐药情况,本文鉴定了鸡蛋生产链中分离得到的111株沙门氏菌(Salmonella)血清型,并测定了抗生素和消毒剂对沙门氏菌的最小抑菌浓度(Minimum inhibitory concentrations, MICs),检测了其对抗生素和消毒剂的耐药基因的表达情况。研究结果表明,沙门氏菌对甲氧苄啶(Trimethoprim, TMP)耐药率最高(N=100,P=90.09%),对阿莫西林/克拉维酸(Amoxicillin and clavulanate, AMC)、头孢噻呋钠(Sodium ceftiofur, CFS)、庆大霉素(Gentamicin, CN)敏感。沙门氏菌共产生6种不同的耐药谱型,TMP是最主要的耐药谱型(N=36,P=32.43%),52.25%的菌株(N=58)具有多重耐药性。苯扎氯铵(Benzalkonium chloride, BC)与氯化十六烷基吡啶(Cetylpyridinium chloride, CPC)对沙门氏菌的MIC的范围分别为：8~128 μg/mL、8~256 μg/mL。相对于质控菌株Escherichia coli ATCC10536,101株沙门氏菌对BC和CPC同时具有较高的耐药性(P=90.99%),109株沙门氏菌对抗生素和消毒剂具有共同耐药性(P=98.20%)。抗生素耐药基因检出率最高为blaTEM(N=49, P=44.14%),未检测出qnrA、qnrB、qepA基因,仅检测出qacEΔ1消毒剂耐药基因(N=63, P=56.76%)。抗生素耐药基因sul1和消毒剂耐药基因qacEΔ1具有显著相关性(P<0.01)。S. Derby对TMP、土霉素(Oxytetracycline, OTC)、阿莫西林(Amoxicillin, AML)、环丙沙星(Ciprofloxacin, CIP)同时表现较高的耐药性,S. Derby检出了11种抗生素耐药基因,消毒剂耐药基因qacEΔ1的检出率为81.25%(N=52)。鸡场中养殖内环境沙门氏菌对抗生素和消毒剂的耐药率以及耐药基因检出率均高于养殖外环境,鸡蛋包装、储存及销售等环节中沙门氏菌耐药率及耐药基因检出率均较高。由此可见,鸡蛋生产链中沙门氏菌对抗生素、消毒剂耐药性较严重,且存在共同耐药的现象。因此,需要进一步规范防控鸡场中沙门氏菌,规范抗生素和消毒剂的使用以及加强鸡蛋生产链条中卫生安全的监管。     

Antibiotic resistance and genotyping of clinical group B Salmonella isolated in Accra,Ghana

AIMS: The purpose of this study was to investigate the antibiotic resistance and clonal lineage of serogroup B Salmonella isolated from patients suspected of suffering from enteric fever in Accra, Ghana. METHODS AND RESULTS: Serogroup B Salmonella were isolated from blood (n=28), cerebral spinal fluid (CSF) (n=1), or urine (n=2), and identified based on standard biochemical testing and agglutinating antisera. Isolates were examined for their susceptibility to ampicillin, chloramphenicol, tetracycline and trimethoprim-sulfamethoxazole. Most of the isolates could be classified as multiple-drug resistant. Furthermore, the genetic location of resistance genes was shown to be on conjugative plasmids. Genetic fingerprinting by plasmid profiling, enterobacterial repetitive intergenic consensus (ERIC)-PCR, and repetitive element (REP)-PCR were performed to determine the diversity among the isolates. Plasmid profiling discriminated five unique groupings, while ERIC-PCR and REP-PCR resulted in two and three groupings, respectively. CONCLUSIONS: A high rate of antibiotic resistance was associated with the Salmonella isolates and the genes responsible for the resistance are located on conjugative plasmids. Also, there appears to be minimal diversity associated with the isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: As a result of the increasing antibiotic resistance among bacteria of all genera, surveys to monitor microbial populations are critical to determine the extent of the problem. The inability to treat many infectious diseases with current antibiotic regimens should prompt the medical community to be more prudent with its antibiotic use.     

Prevalence of Salmonella enterica serovars and genovars from chicken carcasses in slaughterhouses in Spain

AIMS: To determine the prevalence of Salmonella enterica serovars in chicken carcasses in slaughterhouses in Spain and to examine genotypic relations among these serovars. METHODS AND RESULTS: A total of 336 chicken carcasses were collected from six slaughterhouses in Northwestern Spain. Salmonellae were isolated (ISO-6579-1993), serotyped, phage-typed, ribotyped and antibiotyped against 20 antibiotics. Salmonella strains were detected in 60 (17.9%) carcasses. Isolates belonged to nine different serotypes, with Salm. Enteritidis being the most common. Three strains (5%) were resistant to one antibiotic and 24 (40%) were multi-resistant (to more than one antibiotic). The most frequently encountered resistances were to sulphamides, fluoroquinolones and tetracycline. Ribotyping was able to differentiate isolates of the same serotype and phage type. CONCLUSIONS: The Salmonella serotypes and phage types detected are among those most frequently associated with human diseases in Spain. The large percentage of antimicrobial resistant strains is a matter for concern. A high genetic relationship between strains from different slaughterhouses was found. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides detailed information about Salmonella isolates from poultry in Spain. It emphasizes the importance of controlling this pathogen in poultry products, and suggests the need for more prudent use of antibiotics.     

Characterization of antimicrobial susceptibility and virulence genes of Salmonella serovars collected at a commercial turkey processing plant

Aims:  To determine the antimicrobial susceptibility profiles, distribution of class 1 integrons, virulence genes and genes encoding resistance to tetracycline ( tetA , tetC , tetD and tetE ) and streptomycin ( strA , strB and aadA1 ) in Salmonella recovered from turkeys.
Methods and Results:  The antimicrobial susceptibility of 80 isolates was determined using National Antimicrobial Resistance Monitoring System. The distribution of resistance genes, class 1 integrons and virulence genes was determined using PCR. Resistances to tetracycline (76·3%) and streptomycin (40%) were common. Sixty-two (77·5%) isolates displayed resistance against one or more antimicrobials and 33 were multi-drug resistant. tetA was detected in 72·5% of the isolates, while tetC , tetD and tetE were not detected. The strA and strB genes were detected in 73·8% of the isolates. Two isolates possessed class 1 integrons of 1 kb in size, containing the aadA1 gene conferring resistance to streptomycin and spectinomycin. Fourteen of the virulence genes were detected in over 80% of the isolates.
Conclusions:  This study shows that continuous use of tetracycline and streptomycin in poultry production selects for resistant strains. The Salmonella isolates recovered possess significant ability to cause human illness.
Significance and Impact of the Study:  Information from this study can be employed in guiding future strategies for the use of antimicrobials in poultry production.     

Prevalence and antimicrobial susceptibility of Salmonella spp. isolates from US cattle in feedlots in 1999 and 2000

AIMS: Faecal samples from cattle in US feedlots were evaluated for the presence of Salmonella. When Salmonella isolates were recovered the antimicrobial resistance patterns were determined. METHODS AND RESULTS: Faecal samples were collected from pen floors in 73 feedlots in 12 states during the period from October 1999 to September 2000. Pens of cattle selected for sampling were those that had been in the feedlot for the shortest period of time, the longest period of time and a randomly selected pen from the remaining pens. Faecal samples were cultured for Salmonella spp. and all Salmonella isolates were categorized by serotype. The susceptibilities of all isolates were determined using a panel of 17 antimicrobials. Overall, 6.3% (654/10,417) of the samples cultured positive for Salmonella spp. and 22.2% (94/422) of pens and 50.7% (37/73) of feedlots had one or more positive samples. There was little difference in the proportion of positive samples from short-fed (6.1%, 212/3482), random (6.4%, 217/3400) and long-fed (6.4%, 224/3485) pens of cattle. One of two pens of cattle that could not be attributed to a pen type had a single positive sample (2.0%, 1/50). Samples collected during the period of April to June (6.8%, 209/3054) and July to September (11.4%, 286/2500) were more likely to be positive than those collected during October to December (4.0%, 73/1838) and January to March (2.8%, 86/3025). The most common serotypes of Salmonella were dissimilar from those that are typically seen in human illness and cattle illness. The majority of isolates (62.8%, 441/702) were sensitive to all of the antimicrobials tested. Resistance was most frequently observed to tetracycline (35.9%, 252/702) followed by streptomycin (11.1%, 78/702), ampicillin (10.4%, 73/702) and chloramphenicol (10.4%, 73/702). Multiple resistance (resistance to > or =2 antimicrobials) was observed for 11.7% (82/702) of the isolates. CONCLUSIONS: Salmonella was isolated at low frequency from faeces of feedlot cattle and the serotypes were not those commonly associated with human illness. In addition most of the Salmonella isolates were sensitive to all the antimicrobials tested. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributes to understanding the ecology of Salmonella in cattle feedlots and the prevalence of resistance among potential food-borne pathogens.     

Molecular characterization of antimicrobial resistance in Salmonella isolated from animals in Japan

Aims:  To investigate the prevalence of integrons and antimicrobial resistance genes in Salmonella recovered from animals in Japan.
Methods and Results:  Forty-eight out of ninety-four (51·1%) Salmonella isolates showed multidrug resistance phenotypes and harboured at least one antimicrobial resistance gene. Twenty-two out of forty-seven (46·8%) Salmonella enterica serovar Typhimurium that were multidrug-resistant were of definitive phage type DT104. Class 1 integrons were identified in 34/94 isolates (36·2%): 21 isolates containing two gene cassettes, aadA2 and bla _PSE–1, and 13 containing one gene cassette, aadA1 , aadA2 or bla _PSE–1. Class 2 integrons containing estX - sat2 - aadA1 gene cassettes were only identified in Salmonella Enteritidis. The β-lactamase-encoding gene, bla _TEM, was only detected in S. Typhimurium. The plasmid-mediated quinolone resistance gene, qnrS1 , was identified in S. Typhimurium and Salmonella Thompson.
Conclusions:  Our results characterized integrons and antimicrobial resistance genes in Salmonella of animal origin. To the best of our knowledge, this is the first report of qnrS in Salmonella from Japan and also the first report of qnrS in S . Thompson.
Significance and Impact of the Study:  Little is known about the molecular basis of antimicrobial resistance in Salmonella isolated from animals. This study provides useful data on the incidence of integrons and resistance genes in Salmonella of animal origin.     

Chemerin induces lipolysis through ERK1/2 pathway in intramuscular mature adipocytes of dairy bull calves

The adipokine Chemerin has been reported to regulate differentiation and metabolism of adipocytes, but the mechanism underlying lipolysis is still largely unknown. The purpose of this study was to explore whether ERK1/2 pathway is involved in regulating Chemerin during bovine intramuscular mature adipocyte lipolysis. Intramuscular mature adipocytes of dairy bull calves were cultured in vitro and were treated with Chemerin or U0126, which is an inhibitor of ERK1/2 pathway. The results showed that TG content in cells was significantly decreased, glycerol and free fatty acid were significantly increased in cell culture media, and the expression of phosphorylated ERK1/2 in cells was increased in Chemerin-treated group, suggested that ERK1/2 pathway was involved in regulation of lipolysis by Chemerin. In addition, the expression of lipolytic-related critical factors ATGL, HSL, LPL, PPARα, UCP3, and CPT1 were upregulated, but the expression of adipogenic key factors, including PPARγ and C/EBPα were downregulated by Chemerin. Interestingly, all the effects of Chemerin on genes expression in intramuscular mature adipocytes or fat tissue were inhibited by U0126, showed that the function of Chemerin to promote adipose decomposition will be significantly weakened if the ERK1/2 pathway is suppressed, and confirmed that ERK1/2 pathway is involved in mediate Chemerin-enhanced lipolysis. In conclusion, the study demonstrated that Chemerin induce intramuscular mature adipocytes lipolysis through activation of the ERK1/2 pathway. Our research at least provide partial mechanisms of Chemerin on lipolysis and deposition of intramuscular fat tissue of dairy bull calves.     

Phenotypic evidence for inducible multiple antimicrobial resistance in Salmonella choleraesuis

Multiple antimicrobial resistance (MAR) in Salmonella choleraesuis is becoming a major concern. It has been demonstrated that a MAR phenotype can be induced in Escherichia coli and other members of the Enterobacteriaceae by exposing the isolates to salicylates, various antimicrobials, or organic solvents used to combat and control bacterial infection. Therefore the purpose of the present study was to determine whether this marA-associated MAR-phenotype is inducible in S. choleraesuis. Isolates used in the present study were able to withstand toxic effects of the organic solvent cyclohexane naturally, or following exposure to the inducing compounds salicylate, tetracycline, or chloramphenicol. All isolates possessed fragments of marA with the predicted size of 408 bp when amplified using marA-specific primers by PCR. The resulting PCR products that were sequenced revealed that amplified S. choleraesuis marA was 99% and 85% homologous to the published Salmonella typhimurium and E. coli marA sequences respectively. Minimum inhibitory concentrations of tetracycline (P<0.08), chloramphenicol (P<0.001), rifampin (P<0.08), and nalidixic acid (P<0.001) against cyclohexane-tolerant mutants were significantly increased when compared with wild-type S. choleraesuis. Northern hybridization signals for both marA and acrB were increased in the induced isolates when compared to uninduced controls while soxS expression did not change between induced and uninduced cultures. The results suggest that marA is present in S. choleraesuis and a MAR-phenotype is inducible in S. choleraesuis presumably due to the overexpression of marA and acrB and not to the overexpression of soxS.