Rates of resistance of mastitis pathogens from cows in Switzerland

1215 strains of bacteria isolated from cows suffering from acute or chronic clinic mastitis were tested in the antibiogram according to the Kirby-Bauer method. The germ spectrum included: 304 DNase-positive strains of Staphylococci 304 DNase-negative strains of Staphylococci 304 CAMP-negative strains of Streptococci 303 Strains of Enterobacteriaceae The antibiotics selected were Penicillin, Cloxacillin, Neomycin und Gentamicin, then the combinations Penicillin/Neomycin, Cloxacillin/Gentamicin and Nafcillin/Penicillin/Dihydrostreptomycin. Gentamicin showed excellent action against Staphylococci and Enterobacteriaceae. Further on, the present investigations show, that the combination Cloxacillin/Gentamicin is fully effective in the gram-positive spectrum (against Streptococci and Staphylococci), as in the gram-negative spectrum (against E. coli).     

我国奶牛乳房炎无乳链球菌抗生素耐药性研究

为了查明我国奶牛乳房炎无乳链球菌对抗生素耐药情况,指导临床合理用药,从我国部分地区奶牛场采集的临床型奶牛乳房炎病乳中分离鉴定出无乳链球菌115株,采用K-B纸片法测定了这些菌株对抗生素的耐药情况。结果表明,无乳链球菌对目前临床上使用的大部分抗生素,如头孢唑啉、头孢噻肟、丁胺卡那霉素、卡那霉素、庆大霉素、四环素、强力霉素、麦迪霉素、林可霉素、氟苯尼考、多黏菌素B、环丙沙星、氟哌酸、氨苄青霉素/舒巴坦、头孢噻肟/棒酸和头孢他啶/棒酸均比较敏感;但对青霉素G、氨苄青霉素、链霉素、恩诺沙星、阿莫西林/棒酸和复方新诺明等有一定耐药性,其耐药率达50%～100%。     

奶牛乳房炎抗生素防治失败原因探讨

奶牛乳房炎主要由病原微生物引起 ,为奶牛常见病 ,也是造成奶业经济损失最为严重的疾病[1] 。据报道 ,在美国 1 1 0 0万头泌乳奶牛中有约 5 0 %患有乳房炎 ,日本平均乳房炎患病率为 45 .1 % [2 ] ;我国奶牛乳房炎发病率更高 ,孙福先 ( 1 996)、萧乾庆( 1 997)、杨章平 ( 1 998)     

Prevalence of bovine subclinical mastitis and antibiotic susceptibility patterns of major mastitis pathogens isolated in Unguja island of Zanzibar,Tanzania

A cross-sectional study was conducted between January and July 2014 in Unguja island of Zanzibar to establish prevalence of subclinical mastitis (SCM) in smallholder dairy cows and patterns of antibacterial susceptibility of major mastitis pathogens isolated. A total of 416 dairy cows from 201 farmers were randomly selected from three districts of Unguja Island to participate in the study. Questionnaire interview, field observation, individual cow examination, California Mastitis Test (CMT) and bacteriological examination were carried out. Kirby-Bauer disc diffusion technique was used to test drug sensitivity for common bacteria isolated. Based on CMT results, the overall prevalence of SCM was 28.6, 48.8 and 64.7% at quarter, cow and farm level, respectively. Prevalence of bacterial infection was recorded at 42.9, 70.9 and 78.6% at quarter, cow and farm examined, respectively. The common bacteria isolated included Staphylococcus aureus (36.8%), Pseudomonas aeruginosa (17.8%), Staphylococcus epidermidis (16.1%), Klebsiella spp. (9.5%), Micrococcus spp. (6.3%) and Escherichia coli (4.9%). In conclusion, findings of this study demonstrated high level of subclinical mastitis at farms, cows and quarters levels with both contagious and environmental bacterial pathogen involved. Therefore, efforts should be directed to the decreased subclinical mastitis by improving sanitary measures and proper milking practice.     

Prevalence and antibiotic resistance of mastitis pathogens isolated from dairy herds transitioning to organic management

Changes in udder health and antibiotic resistance of mastitis pathogens isolated from dairies upon conversion from conventional to organic management over a 3-year period was studied. Coagulase-negative staphylococci (CNS) were the most prevalent mastitis pathogens isolated. CNS were significantly less resistant to β-lactam antibiotics when isolated from milk after the herd transitioned to organic management. Cessation of the use of antimicrobial therapies in dairies in combination with organic management could lead to a reduction in the antimicrobial resistance of mastitis pathogens.     

检测奶牛乳房炎主要病原菌的多重PCR方法的建立与应用

本试验针对金黄色葡萄球茵、无乳链球菌、大肠杆菌3种主要的乳房炎病原茵设计了3对特异性引物,建立了多重PCR检测方法.结果表明,本方法的特异性为100%,最低检测浓度为1.25×103cfu/mL,充分表明该方法具有快速,准确和特异的优点.     

江苏地区奶牛乳房炎凝固酶阴性葡萄球菌耐药基因分析

对江苏地区多个奶牛场采样,并对奶牛乳房炎(Bovine mastitis,BMT)检测阳性奶样进行病原菌分离、鉴定。对分离出的凝固酶阴性葡萄球菌(Coagulase-negative Staphylococci,CNS)分别进行20种药物的敏感性试验和相关耐药基因检测。结果显示,BMT检测阳性的奶样有610份,528份检出细菌,共检出细菌802株,鉴定为CNS的217株;大部分CNS对万古霉素、左氟沙星、氯霉素、磷霉素、呋喃妥因敏感,对青霉素、大观霉素具有较强耐药性;CNS对β-内酰胺类(blaZ)、大环内酯类(ermC)和四环素类(tetK、tetM、tetL、tetO)耐药基因的检出率分别为29.95%、2.30%、24.88%、1.84%、1.84%和0.00%。     

Molecular types and antibiotic resistance of Staphylococcus aureus isolates from bovine mastitis in a single herd in China

The molecular diversity, antibiotic resistance patterns and presence of resistance genes were determined in Staphylococcus aureus isolates from cases of bovine mastitis in a dairy cattle herd in China. Multiple locus variable number tandem repeat analysis was used for molecular typing. Resistance was determined through minimum inhibitory concentrations and resistance genes were detected by PCR. There was low molecular diversity; one predominant strain (type I) accounted for the majority of cases of S. aureus mastitis in the herd and this strain had a high frequency of resistance to penicillin and tetracycline. The most prevalent resistance genes were blaZ, ermC and tetM.     

Characterization of enterotoxigenic Staphylococcus aureus strains isolated from bovine mastitis in north-east Switzerland

Thirty-four strains of enterotoxin-producing Staphylococcus aureus obtained from milk samples of 34 dairy cows suffering from mastitis from 34 different farms in north-east Switzerland were identified and further characterized by pheno- and genotypic methods. This included the identification of staphylococcal enterotoxin (SE) types, an antibiotic resistance testing, the appraisal of hemolysis, the egg yolk reaction, the detection of the clumping factor and protein A by means of a latex agglutination, the PCR amplification of a S. aureus specific part of the gene encoding the 16S-23S rRNA "intergenic spacer" region and a species specific part of the 23S rRNA-gene, the PCR amplification of the clumping factor (clfA) gene, the X region and the IgG-binding region of the protein A (spa) gene, the coagulase (coa) gene and additionally a macrorestriction analysis of the chromosomal DNA. Within the 26 cultures which formed a single SE, there were 23 SEC- and three SED-formers. Eight cultures were SEAD formers. It was remarkable that 22 SEC formers were also positive for TSST-1. Eighteen of the 23 SEC-formers could be classified as being of the same phenotype. Most of the cultures of one enterotoxin type also showed a great uniformity in the size and number of repeats of the X region as well as in the size of the IgG-binding region of protein A gene and in the size of the coagulase gene. Macrorestriction analysis revealed 11 PFGE patterns. These were in part only different from each other in a few fragments and thus displayed close clonal relations. The results of the present investigation show that a broad distribution of identical or closely related enterotoxin-producing S. aureus clones seem to contribute to the bovine mastitis problem in north-east Switzerland.     

Prevalence of verotoxin-producing Escherichia coli (VTEC) in bovine coli mastitis and their antibiotic resistance patterns.

Between December 1996 and October 1997, milk samples from a total of 145 cows with coli mastitis were screened for the presence of verotoxin-producing E. coli (VTEC). VTEC were found in four (2.8%) out of the 145 samples. The four isolated strains proved to be verotoxin (VT) 1-, VT2- or VT1- and VT2-positive. However, no strain contained all three virulence factors tested. Further strain characterization was carried out by serotyping as well as by resistance pattern analysis.     

Non-oxidative antibacterial activity of bovine neutrophil granule proteins towards mastitis pathogens

Acid extracts of bovine neutrophil granules displayed potent antibacterial activity towards a number of mastitis pathogens in vitro. Killing of pathogens by acid extractable granule protein was dependent on incubation time, protein concentration, bacterial cell load, pH and ionic strength. Gram-negative and Gram-positive organisms showed variable sensitivity to granule extract. Strains of Staphylococcus aureus were the most resistant of tested organisms to granule extract. Gram-negative organisms were neither consistently more nor less sensitive than Gram-positive organisms. Maximal killing of Gram-positive pathogens, after 30 minutes exposure to granule extract at 37 degrees C, occurred between pH 7.0 and 8.0. The Gram-negative organism Escherichia coli B117 was more sensitive to neutrophil granule extract at pH 5.0.     

The major bovine mastitis pathogens have different cell tropisms in cultures of bovine mammary gland cells

We previously showed that Staphylococcus aureus cells adhered mainly to an elongated cell type, present in cultures of bovine mammary gland cells. Moreover, we showed that this adhesion was mediated by binding to fibronectin. The same in vitro model was used here, to study adhesion of other important mastitis pathogens. Like the S. aureus strains, the Streptococcus dysgalactiae strains adhered mainly to elongated cells, which seemed to be mediated by fibronectin binding. In contrast, Streptococcus uberis strains adhered mainly to cubic cells. Since the cubic cells did not express fibronectin and S. uberis cells bound fibronectin less efficiently, the adhesion of S. uberis cells was independent of fibronectin binding. Streptococcus agalactiae strains adhered poorly to both cell types. The specificity and efficiency of adhesion of Escherichia coli strains was strongly strain dependent. None of the S. agalactiae and E. coli strains tested was able to bind fibronectin efficiently. The results suggest that the different mastitis pathogens have different target cell specificities and use different mechanisms to adhere to cells of the bovine mammary gland.     

Isolation of L-form variants after antibiotic treatment in Staphylococcus aureus bovine mastitis

An unstable L-form of Staphylococcus aureus was identified in milk samples from 3 quarters of 2 cows after treatment with cloxacillin. Milk samples incubated on standard 5% blood agar plates were culture-negative for 7 to 30 days after treatment, but S aureus was reisolated in 80% of 66 samples by additional culturing on enriched L-form media when incubated in 10% CO2 at 37 C. The organism was identified at various phases of reversion of L-form agar and was confirmed on transfer to blood agar plates.     

Bactericidal potency and mechanistic specificity of neutrophil defensins against bovine mastitis pathogens

Two neutrophil defensins, rabbit peptides NP-1 and NP-5, were examined for their in vitro antibacterial activity against a panel of Gram-positive and Gram-negative organisms isolated from mastitic cows. Incubation for 60 minutes with 5 micrograms/ml of NP-1 in 10 mM sodium phosphate buffer resulted in substantial killing (greater than 95%) of all organisms tested. Although NP-5 was virtually inactive under these same conditions, supplementation of the incubation mixture with dilute nutrient media sensitized otherwise resistant organisms to this defensin. For both NP-1 and NP-5, bactericidal activity was dependent on time and concentration. Our findings demonstrate that the spectrum of defensin bactericidal activities include clinically important bovine pathogens. Further, the data demonstrate that NP-1 and NP-5, peptides which are homologous in 18 of their 33 residue positions, possess distinct mechanisms of action.     

A proposal of interpretive criteria for cefoperazone applicable to bovine mastitis pathogens

The correct assessment of mastitis pathogens for their susceptibility/resistance to cefoperazone is currently hampered by the lack of harmonized test conditions and interpretive criteria. The aim of this study was to provide a proposal for clinical breakpoints of cefoperazone which are applicable to Staphylococcus aureus, coagulase-negative staphylococci, Escherichia coli, Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis from cases of bovine mastitis and better reflect the situation in the bovine udder than breakpoints adopted from human medicine. For this, pharmacological data and clinical efficacy data of the documents submitted for approval of cefoperazone have been revisited. In addition, 1086 bacterial pathogens of the aforementioned six species/groups collected in Germany and in the USA during recent years were tested in parallel for their cefoperazone MICs and the zone diameters using a 75 μg disk. Subsequently, MICs were plotted against zone diameters. Based on the pharmacological data, the clinical efficacy and the microbiological data, a proposal was made for veterinary-specific breakpoints which classify members of the aforementioned species/groups as (a) susceptible to cefoperazone when their MIC is ≤ 2 μg/ml and their zone diameters are ≥ 27 mm (staphylococci or E. coli) or ≥ 21 mm (streptococci), (b) intermediate when their MIC is 4 μg/ml and their zone diameters are 22-26 mm (staphylococci or E. coli) or 16-20mm (streptococci), and (c) resistant when their MIC is ≥ 8 μg/ml and their zone diameters are ≤ 21 mm (staphylococci or E. coli) or ≤ 15 mm (streptococci).     

Antibacterial activity of bovine lactoferrin hydrolysate against mastitis pathogens and its effect on superoxide production of bovine neutrophils

Antibacterial activity of bovine lactoferrin hydrolysates (LFH) on microorganisms isolated from bovine mastitis, and superoxide (O(2)(-)) production of bovine neutrophils were evaluated. Antibacterial effects of LFH were measured in vitro against Staphylococcus aureus, coagulase-negative staphylococci, Streptococci, Enterococci, Escherichia coli, Klebsiella pneumoniae, yeast-like fungi and Prototheca zopfii isolated from clinical cases of bovine mastitis. To compare susceptibilities against LFH, minimal inhibitory concentration (MIC) values were determined by a micro-plate assay method. Most organisms were sensitive to LFH. Prototheca zopfii was highly sensitive to LFH; the growth of the microorganism was inhibited completely even at 1 mug/ml. Staphylococcus aureus and Escherichia coli were resistant to LFH. The production of O(2)(-) by bovine neutrophils was used to evaluate the effect of LFH administration on functional activity. Increase in O(2)(-) production by bovine neutrophils occurred upon addition of LFH to neutrophils. These results demonstrate that LFH possesses antibacterial activity against pathogens that cause mastitis and activates neutrophil superoxide production.