牛源金黄色葡萄球菌凝固酶基因的分型

为了了解我国不同地区奶牛场金黄色葡萄球菌基因型的分布情况,采用凝固酶基因（Coa）扩增的分型方法,对在奶牛乳房炎奶样中所分离到的26株金黄色葡萄球菌进行了分型。结果显示,26株金黄色葡萄球菌共分为5个基因型,黑龙江省分离株以PCR 3型为主（10/16）。发现在同一牛群中的分离株可以存在相同的基因型,也可以存在不同的基因型;而不同牛群中的分离株也可属于同一基因型。这种结果提示,在某个或某些牛群中存在某一金黄色葡萄球菌的流行型,但也可能存在多个金黄色葡萄球菌的克隆。     

Coagulase gene polymorphisms detected by PCR in Staphylococcus aureus isolated from subclinical bovine mastitis in Turkey

The genetic relatedness of coagulase (coa) positive Staphylococcus aureus isolated from cows with subclinical mastitis in Turkey was investigated by polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) analysis. Among 700 milk samples positive in the California Mastitis Test (CMT), species specific PCR identified 200 (28.6%) isolates as S. aureus and 161 (80.5%) of these isolates were positive for the 3' end of the coa gene by PCR. Most isolates (n=135, 83.9%) produced a single band on coa PCR, with molecular sizes ranging from 500 to 1400bp, whereas a small number of isolates (n=26, 16.1%) yielded two amplification products. Coa RFLP analysis using AluI and Hin6I revealed 23 and 22 band patterns, respectively. The detection of double bands by coa PCR, previously reported in human isolates, suggests that milking personnel can play a role in the transmission of S. aureus.     

Molecular typing of enterotoxigenic Staphylococcus aureus isolated from bovine mastitis in Korea

One hundred and sixty-six Staphylococcus aureus isolates from mastitic milk samples from different cows on 26 farms were investigated for staphylococcal enterotoxins(SEs) and toxic shock syndrome toxin-1(TSST-1) by polymerase chain reaction(PCR) and reverse passive latex agglutination assay(RPLA). SEs and the TSST-1 gene were detected in thirty-seven isolates based on a multiplex PCR; SEA was detected in 32 isolates, SEB in 3 isolates, SEC in 1 isolate, and SEA and the TSST-1 gene in 1 isolate. Of the 37 enterotoxigenic isolates, thirty-three isolates were enterotoxigenic according to RPLA, where 29 isolates produced SEA, 3 isolates produced SEB, and 1 isolate produced SEC. The enterotoxin-producing S. aureus isolates were further characterized by pulsed-field gel electrophoresis(PFGE). A macrorestriction analysis revealed 11 PFGE patterns. Among the 33 enterotoxigenic S. aureus isolates, 45.4% exhibited the same PFGE pattern I. Accordingly, although the enterotoxin-producing S. aureus isolates from bovine mastitis were genetically diverse, 1 common genotype prevailed on the farms, indicating that PFGE pattern I isolates may be the most disseminated in Korea.     

Phage conversion of exfoliative toxin A in Staphylococcus aureus isolated from cows with mastitis

An exfoliative toxin produced by Staphylococcus aureus is the causative agent of staphylococcal scalded-skin syndrome (SSSS) in young children. Recently, we reported that only few isolates of S. aureus from bovine mastitis contained the eta gene encoding exfoliative toxin A (ETA) and produced ETA in vitro. In this study, we isolated temperate phages from two ETA-positive bovine isolates of S. aureus by treatment with mitomycin C. Polymerase chain reaction (PCR) assay of the phage genomes suggested that the temperate phages carried the structural gene for ETA. Moreover, the nucleotide sequence analysis of the PCR products revealed that the eta gene was located very close to an amidase gene on the phage genomes. The nucleotide sequence for the amidase gene of the bovine phage (bovine phi ETA) differed at nine positions from that of the amidase gene of phi ETA from a human isolate reported by Yamaguchi et al. [Mol. Microbiol. 38 (2000) 694], suggesting that eta-converting phages are heterogeneous. Bovine phi ETA had a head with a hexagonal outline and a non-contractile and flexible tail. Bovine phi ETA was able to lysogenize ETA-negative bovine isolates of S. aureus, and the lysogenized S. aureus isolates had the ability to produce ETA. These results suggest the possibility of horizontal transmission of the eta gene by temperate bacteriophages among bovine isolates of S. aureus.     

新疆部分地区奶牛乳房炎金黄色葡萄球菌荚膜多糖分型的研究

为调查新疆奶牛乳房炎金黄色葡萄球菌主要流行血清型,本研究采用玻板凝集和双重PCR方法对不同地区奶牛乳房炎乳样中分离的117株金黄色葡萄球菌进行血清分型.结果表明荚膜多糖5型占10.26％(12/117),8型占13.68％(16/117),336型占64.96％(76/117),未分型菌株占11.11 ％(13/117).该研究为新疆奶牛乳房炎金黄色葡萄球菌疫苗菌株的筛选提供了依据.     

Phage typing of strains of Staphylococcus aureus from staphylococcal mastitis of dairy cows]

Of 62 strains of Staphylococcus aureus, isolated from milk samples of dairy-cows with staphylococcic mastitis from five agricultural enterprises, 68 p. c. could be typified as bacteriophages. In 42 typified strains, altogether 17 different phage pictures were found, in which case, in the different agricultural enterprises, the number of phage picture fluctuated between 1 and 8. Phage picture 42 D was found and dominated in all the agricultural enterprises. Of the typified strains, 76% could be ranked only into one phage group and 24% in the "M" group (= miscellaneous). Of 32 typified strains, 56.3% belonged to phage group IV, 25% to group III, 12.5% to group I, and 6.2% to group II.     

牛源金黄色葡萄球菌16SrRNA分子鉴定与随机多态性扩增分型

利用16S rRNA保守序列对引起内蒙古地区奶牛乳房炎的金黄色葡萄球菌进行分离鉴定,并且建立引物随机多态性扩增(RAPD)体系对金黄色葡萄球菌分离株进行基因分型研究.结果表明,共分离出金黄色葡萄球菌35株,RAPD结果显示这35株菌均可得到清晰的RAPD指纹图谱,扩增产物在1～8条带之间,产物大小在350～4 500bp之间.菌株分为6个基因型,Ⅳ型为该地区的流行优势菌群.不同牛场各基因型菌株分布有明显差异,这与牛场的环境和病原菌在牛场间流行传播情况有关.研究结果为地区性奶牛乳房炎的防治及新疫苗的研制提供了可靠的理论依据.     

Characteristics of Staphylococcus aureus from subclinical bovine mastitis in Brazil

A total of 127 Staphylococcus aureus strains isolated from milk samples of cows with subclinical bovine mastitis was examined for biotype, phage pattern, in-vitro antibiotic susceptibilities and ability to produce enterotoxins. The majority of the strains showed features consistent with bovine rather than human origin. All strains were sensitive to the antibiotics tested, except penicillin and streptomycin. Enterotoxigenicity was observed in 6 (4.7%) strains and only enterotoxins A and C were produced.     

In vitro susceptibility of Staphylococcus aureus strains isolated from cows with subclinical mastitis to different antimicrobial agents

Sensitivity to commercial teat dips (nonoxinol-9 iodine complex and chlorhexidine digluconate) of 56 Staphylococcus (S.) aureus strains isolated from quarter milk samples of various German dairy herds treated with different teat dipping schemes was investigated in this study. The minimum inhibitory concentration was determined using a broth macrodilution method according to the German Veterinary Association guidelines. The main objective of the current study was to induce in vitro resistance induction of S. aureus to chemical disinfectants. Ten different strains were repeatedly passed ten times in growth media with sub-lethal concentrations of disinfectants. Nine strains showed a significant reduction in susceptibility to the nonoxinol-9 iodine complex but only one strain developed resistance to chlorhexidine digluconate. Stability of the acquired resistance was observed in all S. aureus strains adapted to the nonoxinol-9 iodine complex and chlorhexidine digluconate. In contrast, simultaneous resistance to different antibiotics was not observed in any of the ten investigated S. aureus strains. However, the isolates exhibited a high degree of resistance to penicillin G. Based on these results, resistance of S. aureus to chemical disinfectants may be more likely to develop if the chemicals are used at concentrations lower than that required for an optimal biocidal effect.     

Variation of the agr locus in Staphylococcus aureus isolates from cows with mastitis

Staphylococcus aureus isolates from mastitic cow's milk were examined for production of alpha-hemolysin and protein A and their accessory gene regulator (agr locus) was analyzed. An inverse relationship between alpha-hemolysin and protein A production was found in most of the 76 isolates, suggesting that the isolates tested may be classified into group I (high alpha-hemolysin/low protein A), II (low alpha-hemolysin/high protein A), or III (low alpha-hemolysin/low protein A). The agr locus, which consists of hld, agrB, agrD, agrC, and agrA, was detected in most of the 78 isolates including two reference strains (Wood 46 and Cowan I) by polymerase chain reaction (PCR). When the PCR products for agr locus of 22 isolates from groups I and II were digested with restriction enzyme MboI, seven bands of the expected lengths were recognized in strain Wood 46, but not in the other isolates tested. Nucleotide sequence analysis of PCR products from six isolates revealed that the agr locus sequence of strain Wood 46 corresponded to that of the published sequence data, but the other five isolates from groups I and II diverged at agrB and agrD sequences and thus the deduced amino acid sequences. These variations of agr locus in S. aureus bovine isolates differed from those reported by Ji et al. [Science 276 (1997) 2027].     

Surface properties of Staphylococcus aureus isolated from caprine mastitis

A total of 53 Staphylococcus aureus strains isolated from caprine mastitis were tested for surface hydrophobicity, surface Protein A (SpA), and binding capacity of fibronectin, fibrinogen and type II collagen. Strong positive correlation was found between surface hydrophobicity and SpA, and between surface hydrophobicity and 125I-fibronectin-binding. Regardless of hydrophobicity, the binding of fibrinogen was moderate and type II collagen binding was low. The results indicate that SpA and fibronectin-binding protein contribute to the high relative surface hydrophobicity of S. aureus associated with caprine mastitis.     

Genotyping of Staphylococcus aureus isolated from bovine mastitis.

The present study was designed to comparatively investigate 25 Staphylococcus aureus strains isolated from bovine subclinical mastitis. The S. aureus strains, obtained from six different farms at five locations in one region of Germany, were characterized by phenotypic and genotypic methods. The S. aureus could be identified and further characterized by their cultural, biochemical and hemolytic properties. To analyze the epidemiological relationship the isolates were subjected to DNA fingerprinting by macrorestriction analysis of their chromosomal DNA, by PCR amplification of the gene encoding the 16S-23S rRNA intergenic spacer, by PCR amplification of the gene encoding the IgG binding region and the X region of protein A and by amplifying, and subsequent, digestion of the gene encoding staphylococcal coagulase. The macrorestriction analysis revealed five DNA restriction patterns with DNA patterns I, III and IV occurring in three, four, and three different farms, respectively. In addition, clones with different DNA patterns could be found within one herd. The PCR products for the spacer DNA, the spa gene encoding the X region of protein A and the coa gene encoding coagulase corresponded mostly to the pattern observed by DNA fingerprinting. Amplification of the gene encoding the IgG binding region revealed sizes of 620 bp for 20 of the isolates and 280 bp for four isolates indicating, for the latter, a deletion of segments in this region. These findings show, that single, widely distributed clones seemed to be responsible for cases of bovine subclinical mastitis found in one region of Germany.     

我国部分地区奶牛乳房炎病例中金黄色葡萄球菌血清型及毒力调查

为了解我国奶牛乳房炎病例中金黄色葡萄球菌(S.aureus)流行血清型和不同血清型菌株的毒力情况,本研究采用玻板凝集和双重PCR方法分别对北京、山西、内蒙古、山东、浙江和新疆奶牛乳房炎乳样中分离的191株S.aureus进行血清分型并从各血清型中随机选取100株菌以小白鼠进行毒力测定.结果表明:这些地区牛源S.aureus中,336PS型为流行血清型,占60.2％(115/191),CP8型占19.4％(37/191),CP5型占11.5％(22/191),未分型菌株占8.9％(17/191).毒力测定显示:336PS、CP8和CP5型菌株中强毒力菌株分别占65.1％、62.9％及61.9％,未分型菌株中强毒力菌株为22.2％.该调查为我国奶牛乳房炎S.aureus疫苗菌株的筛选提供了依据.     

Investigation of biofilm production and icaA and icaD genes in Staphylococcus aureus isolated from heifers and cows with mastitis

Biofilm formation and antimicrobial resistance of Staphylococcus aureus are important virulence factors in cases of mastitis in dairy cows. However, few studies have investigated mastitis strains isolated from heifers. Within this context, the objective of the present study was to investigate biofilm formation on Congo red agar, the presence of the icaA and icaD genes by polymerase chain reaction (PCR), and the percentage of in vitro antimicrobial resistance of 110 S. aureus isolates from mammary gland secretions of heifers and cows with mastitis. PCR detected the icaA and icaD genes in 98% and 100% of isolates, respectively. However, only 55.5% of all isolates produced a biofilm on Congo red agar. Antimicrobial susceptibility testing revealed that 47.0% of isolates from heifers and 70.4% of isolates from cows were resistant to at least one of the antimicrobial agents tested. Resistance to penicillin and/or ampicillin was the most frequent (44.5%). These results indicate the need to implement prophylactic and control measures of mastitis for heifers. Heifers and cows can carry resistant strains with the capacity of biofilm production, a fact representing a threat to public health and animal well‐being and generating losses to dairy farmers.     

Antimicrobial drug susceptibility of Staphylococcus aureus isolated from bovine mastitis

Isolates of Staphylococcus aureus (106) from bovine mastitis were tested for susceptibility to antimicrobial agents. beta-lactamase was produced by 69.8 per cent of isolates and 7.5 per cent were resistant to streptomycin (minimum inhibitory concentration more than 32 micrograms/ml). Resistance to other agents was rare. Intrinsic resistance or tolerance to beta-lactam antibiotics was not found.     

Molecular typing of Staphylococcus aureus isolated from cows with mastitis in the east of Poland on the basis of polymorphism of genes coding protein A and coagulase

The aim of this study was to test the diversity of a population of 82 strains of S. aureus isolated from cows with mastitis in the east of Poland. The isolates were typed by analysis of the number of repeats of 24 bp sequences in the X region of protein A (spa) gene and restriction fragment length polymorphism (RFLP) of the coagulase (coa) gene. Twelve different spa types were distinguished. Amplification of region X gave, in 79 cases, one stripe. In a scope of 100-364 bp 10 different products (genotypes) of amplification reaction were defined. For one strain two stripes were obtained and two strains did not contain the spa gene. The most prevalent strains had 10, 11 and 12 repeats of 24 bp sequences, which represented respectively 18%, 30% and 13% of all strains tested. The presence of any strain containing 4 or 9 sequence was not observed. In the case of analysis of the polymorphism of the coagulase gene, 13 different genotypes were identified. The most frequently appearing genotype is genotype C, in which case an amplification product is digested into three DNA fragments: 410, 320 and 160 bp. To this genotype belong 43 strains, which constitute 52% of the examined population. A significant improvement in discriminatory power was observed when results from both genes were analyzed simultaneously. In an analyzed group of 82 strains, 24 genotypes were isolated.     

Serological proteome analysis of Staphylococcus aureus isolated from sub-clinical mastitis

Staphylococcus aureus is the most common aetiologic agent of contagious bovine mastitis. Studies of the molecular epidemiology of S. aureus strongly suggest that some genetic subsets of strains are particularly well adapted for causing infections in cattle. This communication reports the setup of experimental protocols to identify the immunogenic proteins expressed by one of the most common field isolated strain of S. aureus responsible for sub-clinical mastitis cases. The serological proteome analysis (SERPA) approach applied consists of three main steps: two-dimensional electrophoresis-based separation of the proteins contained in field isolated S. aureus extracts enriched for surface proteins, detection of immunogenic spots using anti-serum collected from sub-clinical mastitis cases and identification of antigens by mass spectrometric-based methodologies. The study allowed to identify three immunogenic proteins: DNAase translocase FtsK, ribosomal proteins S1 and a Tell-like protein.     

Characterization of Staphylococcus aureus strains isolated from bovine mastitis in Japan

Fifty-three strains of Staphylococcus aureus isolated from cows affected with mastitis from 21 prefectures in Japan were characterized by phenotypic and genotypic methods. Thirty-three (62.3%) strains showed biotype K-beta+CV:A, coagulase type VI, and sensitivity to bovine phages of group III or IV. These 33 strains could be subdivided into two groups on the basis of the production of staphylococcal enterotoxins (SEs) and on toxic shock syndrome toxin-1 (TSST-1). By pulsed-field gel electrophoresis, the 16 SEC- and TSST-1-producing strains showed similar patterns that differed by only a few fragments, suggesting that they were genetically closely related. Fifteen of 17 non SEC-producing strains which did not produce any other SEs and TSST-1 were genetically different from the SEC-producing strains and showed genetic diversity.     

奶牛乳房炎病例中金黄色葡萄球菌毒素基因的检测

金黄色葡萄球菌是引起奶牛乳房炎的重要病原菌之一，其不仅给奶业生产造成严重损失，而且携带毒素基因的金黄色葡萄球菌在适当环境温度、pH、介质下可产生毒素，影响乳产品质量安全，危害人体健康。金黄色葡萄球菌常产生的、临床意义较大的毒素为肠毒素（SEs）、剥脱毒素（ETs）和中毒休克毒素-1（TSST-1）三类。国外有较多奶牛乳房炎金黄色葡萄球菌毒素基因流行情况的调查报道，国内少有这方面的研究。因此，本文就我国重要奶产地呼和浩特地区奶牛乳房炎金黄色葡萄球菌的sea、seb、sec、sed、see、eta、etb、tst等8种常见毒素基因的流行情况进行调查研究，以期为食源性疾病的监控和分子检测提供科学依据。